Methods and devices for collecting, handling and processing mammary fluid samples for evaluating breast diseases, including cancer

ABSTRACT

Biological samples of mammary fluid or components thereof are obtained using a breast pump device coupled with a solid phase sample collection medium, alternatively facilitated by administering oxytocin to the subject. The breast pump device stimulates expression of mammary fluid and provides for collection of diagnostic samples to evaluate breast disease, including cancer. The biological sample may include whole cells or cellular components, purified or bulk proteins, glycoproteins, peptides, nucleotides or other desired constituents comprising a breast disease marker. Methods, kits and adapter devices relating to the breast pump device are also provided. Yet additional methods, devices, accessories, and materials are provided for laboratory handling and processing of breast fluid samples and for related diagnostic methods.

RELATED APPLICATIONS

[0001] This application claims the priority benefits of U.S. ProvisionalPatent Application No. 60/248,134, filed by Quay on Nov. 13, 2000 andU.S. Provisional Patent Application No. 60/248,136, filed by Quay onNov. 13, 2000. The disclosures of each of the foregoing priorityapplications are incorporated herein by reference in their entirety.

TECHNICAL FIELD OF INVENTION

[0002] The invention relates to methods, devices, and kits for obtainingand assaying biological samples from mammary fluid. More specifically,the invention relates to methods, devices, and kits for obtaining andassaying fluid and cytological samples from the mammary glands of amammalian subject for evaluating, diagnosing and managing breastdisease, including infections, pre-cancerous conditions, cancersusceptibility and cancer.

BACKGROUND OF THE INVENTION

[0003] Breast cancer is by far the most common form of cancer in women,and is the second leading cause of cancer death in humans. Despite manyrecent advances in diagnosing and treating breast cancer, the prevalenceof this disease has been steadily rising at a rate of about 1% per yearsince 1940. Today, the likelihood that a women living in North Americawill develop breast cancer during her lifetime is one in eight.

[0004] The current widespread use of mammography has resulted inimproved detection of breast cancer. Nonetheless, the death rate due tobreast cancer has remained unchanged at about 27 deaths per 100,000women. All too often, breast cancer is discovered at a stage that is toofar advanced, when therapeutic options and survival rates are severelylimited. Accordingly, more sensitive and reliable methods are needed todetect small (less than 2 cm diameter), early stage, in situ carcinomasof the breast. Such methods should significantly improve breast cancersurvival, as suggested by the successful employment of Papinicolousmears for early detection and treatment of cervical cancer.

[0005] In addition to the problem of early detection, there remainserious problems in distinguishing between malignant and benign breastdisease, in staging known breast cancers, and in differentiating betweendifferent types of breast cancers (e.g. estrogen dependent versusnon-estrogen dependent tumors). Recent efforts to develop improvedmethods for breast cancer detection, staging and classification havefocused on a promising array of so-called cancer “markers.” Cancermarkers are typically proteins that are uniquely expressed (e.g. as acell surface or secreted protein) by cancerous cells, or are expressedat measurably increased or decreased levels by cancerous cells comparedto normal cells. Other cancer markers can include specific DNA or RNAsequences marking deleterious genetic changes or alterations in thepatterns or levels of gene expression associated with particular formsof cancer.

[0006] A large number and variety of breast cancer markers have beenidentified to date, and many of these have been shown to have importantvalue for determining prognostic and/or treatment-related variables.Prognostic variables are those variables that serve to predict diseaseoutcome, such as the likelihood or timing of relapse or survival.Treatment-related variables predict the likelihood of success or failureof a given therapeutic plan. Certain breast cancer markers clearly serveboth functions. For example, estrogen receptor levels are predictive ofrelapse and survival for breast cancer patients, independent oftreatment, and are also predictive of responsiveness to endocrinetherapy. Pertschuk et al., Cancer 66:1663-1670, 1990; Parl and Posey,Hum. Pathol. 19:960-966, 1988; Kinsel et al., Cancer Res. 49:1052-1056,1989; Anderson and Poulson Cancer 65:1901-1908, 1989.

[0007] The utility of specific breast cancer markers for screening anddiagnosis, staging and classification, monitoring and/or therapypurposes depends on the nature and activity of the marker in question.For general reviews of breast cancer markers, see Porter-Jordan et al.,Hematol. Oncol. Clin. North Amer. 8:73-100, 1994; and Greiner,Pharmaceutical Tech., May, 1993, pp. 28-44. As reflected in thesereviews, a primary focus for developing breast cancer markers hascentered on the overlapping areas of tumorigenesis, tumor growth andcancer invasion. Tumorigenesis and tumor growth can be assessed using avariety of cell proliferation markers (for example Ki67, cyclin D1 andproliferating cell nuclear antigen (PCNA)), some of which may beimportant oncogenes as well. Tumor growth can also be evaluated using avariety of growth factor and hormone markers (for example estrogen,epidermal growth factor (EGF), erbB-2, transforming growth factor (TGF),which may be overexpressed, underexpressed or exhibit altered activityin cancer cells. By the same token, receptors of autocrine or exocrinegrowth factors and hormones (for example insulin growth factor (IGF)receptors, and EGF receptor) may also exhibit changes in expression oractivity associated with tumor growth. Lastly, tumor growth is supportedby angiogenesis involving the elaboration and growth of new bloodvessels and the concomitant expression of angiogenic factors that canserve as markers for tumorigenesis and tumor growth.

[0008] In addition to tumorigenic, proliferation and growth markers, anumber of markers have been identified that can serve as indicators ofinvasiveness and/or metastatic potential in a population of cancercells. These markers generally reflect altered interactions betweencancer cells and their surrounding microenvironment. For example, whencancer cells invade or metastasize, detectable changes may occur in theexpression or activity of cell adhesion or motility factors, examples ofwhich include the cancer markers Cathepsin D, plasminogen activators,collagenases and other factors. In addition, decreased expression oroverexpression of several putative tumor “suppressor” genes (for examplenm23, p53 and rb) has been directly associated with increased metastaticpotential or deregulation of growth predictive of poor disease outcome.

[0009] Additional representative breast disease markers within thesevarious classes include prostaglandin E2 (PGE2); estrogen-regulatedproteins such as pS2; interleukins (e.g., IL-10); S-100 protein;vimentin; epithelial membrane antigen; prostate specific antigen (PSA);bcl-2; CA15-3 (an aberrant form of polymorphic epithelial mucin (PEM));CA 19-9; mucin core carbohydrates (e.g., Tn antigen and Tn-likeantigens); alpha-lactalbumin; lipidassociated sialic acid (LASA);galactose-N-acetylgalactosamine (Gal-GaINAC); GCDFP-15; Le(y)-relatedcarbohydrate antigen; CA 125; urokinase-type plasminogen activator (uPA)and uPA related antigens and complexes (e.g., LMW-uPA, HMW-uPA, uPAaminoterminal fragment (ATF), uPA receptor (UPAR) and complexes withinhibitors such as PA1-1 and PA1-2); beta-glucuronidase; CD31; CD44splice variants; blood group antigens (e.g., ABH, Lewis, and MN); andgenetic lesions or altered expression levels of CCND1, EMS1, BRCA1 andBRCA2 genes.

[0010] In summary, the evaluation of proliferation markers, oncogenes,growth factors and growth factor receptors, angiogenic factors,proteases, adhesion factors and tumor suppressor genes, among othercancer markers, can provide important information concerning the risk,presence, status or future behavior of cancer in a patient. Determiningthe presence or level of expression or activity of one or more of thesecancer markers can aid in the differential diagnosis of patients withuncertain clinical abnormalities, for example by distinguishingmalignant from benign abnormalities. Furthermore, in patients presentingwith established malignancy, cancer markers can be useful to predict therisk of future relapse, or the likelihood of response in a particularpatient to a selected therapeutic course. Even more specific informationcan be obtained by analyzing highly specific cancer markers, orcombinations of markers, which may predict responsiveness of a patent tospecific drugs or treatment options.

[0011] Methods for detecting and measuring cancer markers have beenrecently revolutionized by the development of immunological assays,particularly by assays that utilize monoclonal antibody technology.Previously, many cancer markers could only be detected or measured usingconventional biochemical assay methods, which generally require largetest samples and are therefore unsuitable in most clinical applications.In contrast, modern immunoassay techniques can detect and measure cancermarkers in relatively much smaller samples, particularly when monoclonalantibodies that specifically recognize a targeted marker protein areused. Accordingly, it is now routine to assay for the presence orabsence, level, or activity of selected cancer markers byimmunohistochemically staining breast tissue specimens obtained viaconventional biopsy methods. Because of the highly sensitive nature ofimmunohistochemical staining, these methods have also been successfullyemployed to detect and measure cancer markers in smaller, needle biopsyspecimens which require less invasive sample gathering procedurescompared to conventional biopsy specimens. In addition, otherimmunological methods have been developed and are now well known in theart which allow for detection and measurement of cancer markers innon-cellular samples such as serum and other biological fluids frompatients. The use of these alternative sample sources substantiallyreduces the morbidity and costs of assays compared to proceduresemploying conventional biopsy samples, which allows for application ofcancer marker assays in early screening and low risk monitoring programswhere invasive biopsy procedures are not indicated.

[0012] For the purpose of breast cancer evaluation, the use ofconventional or needle biopsy samples for cancer marker assays is oftenundesirable, because a primary goal of such assays is to detect thecancer before it progresses to a palpable or mammographically detectabletumor stage. Prior to this stage, biopsies are generallycontraindicated, making early screening and low risk monitoringprocedures employing such samples untenable. Therefore, there is generalneed in the art to obtain samples for breast cancer marker assays byless invasive means than biopsy, for example by serum withdrawal.

[0013] Efforts to utilize serum samples for breast cancer marker assayshave met with limited success, largely because the targeted markers areeither not detectable in serum, or because telltale changes in thelevels or activity of the markers cannot be monitored in serum. Inaddition, the presence of breast cancer markers in serum probably occursat the time of micro-metastasis, making serum assays less useful fordetecting pre-metastatic disease. In contrast, fluid within the mammaryglands themselves is expected to contain much higher and morebiologically relevant levels of breast cancer markers than serum,particularly in view of the fact that 80%-90% of all breast cancersoccur within the intraductal epithelium of these glands. Fluid withinthe breast ducts is expected to contain an assemblage and concentrationof hormones, growth factors and other potential markers comparable tothose secreted by, or acting upon, the surrounding cells of thealveolar-ductal system. Likewise, mammary fluid is expected to containcells and solid cellular debris or products that can be used incytological or immunological assays to evaluate intracellular or cellsurface markers that may not be detectable in the liquid fraction ofmammary fluid.

[0014] Previous attempts to develop non-invasive breast cancer markerassays utilizing mammary fluid samples have included studies of mammaryfluid obtained from patients presenting with spontaneous nippledischarge. In one of these studies, conducted by Inaji et al., Cancer60:3008-3013, 1987, levels of the breast cancer marker carcinoembryonicantigen (CEA) were measured using conventional, enzyme linkedimmunoassay (ELISA) and sandwich-type, monoclonal immunoassay methods.These methods successfully and reproducibly demonstrated that CEA levelsin spontaneously discharged mammary fluid provide a sensitive indicatorof nonpalpable breast cancer. In a subsequent study, also by Inaji etal., Jpn. J. Clin. Oncol. 19:373-379, 1989, these results were expandedusing a more sensitive, dry chemistry, dot-immunobinding assay for CEAdetermination. This latter study reported that elevated CEA levelsoccurred in 43% of patients tested with palpable breast tumors, and in73% of patients tested with nonpalpable breast tumors. CEA levels in thedischarged mammary fluid were highly correlated with intratumoral CEAlevels, indicating that the level of CEA expression by breast cancercells is closely reflected in the mammary fluid CEA content. Based onthese results, the authors concluded that immunoassays for CEA inspontaneously discharged mammary fluid are useful for screeningnonpalpable breast cancer.

[0015] Although the evaluation of mammary fluid has been shown to be auseful method for screening nonpalpable breast cancer in women whoexperience spontaneous nipple discharge, the rarity of this conditionrenders the methods of Inaji et al, inapplicable to the majority ofwomen who are candidates for early breast cancer screening. In addition,the first Inaji report cited above determined that certain patientssuffering spontaneous nipple discharge secrete less than 10 μl ofmammary fluid, which is a critically low level for the ELISA andsandwich immunoassays employed in that study. It is likely that otherantibodies used to assay other cancer markers may exhibit even lowersensitivity than the anti-CEA antibodies used by Inaji and coworkers,and may therefore not be adaptable or sensitive enough to be employedeven in dry chemical immunoassays of small sampbs of spontaneouslydischarged mammary fluid.

[0016] In view of the above, an important need exists in the art formore widely applicable, non-invasive methods and materials to obtainbiological samples for use in evaluating, diagnosing and managing breastdisease including cancer, particularly for screening early stage,nonpalpable breast tumors. A related need exists for methods andmaterials that utilize such readily obtained biological samples toevaluate, diagnose and manage breast disease, particularly by detectingor measuring selected breast cancer markers, or panels of breast cancermarkers, to provide highly specific, cancer prognostic and/ortreatment-related information, and to diagnose and manage pre-cancerousconditions, cancer susceptibility, breast infections and other breastdiseases.

SUMMARY OF THE INVENTION

[0017] It is therefore an object of the present invention to providenon-invasive methods and kits for obtaining biological samples that canbe employed in assays for evaluating, diagnosing and managing breastdisease, particularly cancer.

[0018] It is a further object of the invention to achieve the aboveobject in assay methods and kits that are widely applicable to a broadrange of patients, and that include useful assays and kits for screeningearly stage, nonpalpable mammary tumors.

[0019] It is yet another object of the invention to provide methods andkits that utilize the aforementioned biological samples to evaluate,diagnose and manage breast disease, preferably breast cancer, bydetecting and/or measuring selected breast disease markers such asbreast cancer markers, or panels of breast cancer markers, to providehighly specific prognostic and/or treatment-related information to theclinician.

[0020] The invention achieves these objects and other objects andadvantages that will become apparent from the description which followsby providing non-invasive methods and devices for obtaining biologicalsamples from a mammary organ of a mammalian patient. Through the use ofnovel, specialized breast pump devices of the invention, which arefluidly connected with (i.e., by direct or indirect coupling) a solidphase sample collection medium, the physician can rapidly andnon-invasively collect mammary fluid samples from lactating ornon-lactating female patients without additional intervention. Alternatemethods of the invention for mammary fluid sample collection may involveadministration of oxytocin, or an oxytocin analog, in an amounteffective to stimulate or increase expression of mammary fluid inducedin conjunction with employment of the breast pump device. The oxytocinor oxytocin analog (for example a long-acting oxytocin analog such ascarbetocin) is administered in a manner (e.g., intranasally) and amountsufficient to reach and stimulate a target alveolar-ductal tissue of thebreast, whereby the oxytocin stimulates myoepithelial contraction of thealveolar-ductal tissue to induce or facilitate mammary fluid expression.Alternatively, an intramuscular or intravascular injection of oxytocincan effect the same myoepithelial contraction response as the intranasaladministration route. The amount, timing and/or mode of oxytocinadministration may be adjusted on an individual basis depending on suchfactors as menstrual cycle stage, use of birth control or hormonereplacement therapy, pregnancy history, age of onset of menarche,ethnicity and other factors known to affect an individual's propensityfor breast fluid expression.

[0021] Mammary fluid collection devices of the invention are effectiveto induce mammary fluid expression for sample collection, alone or inconjunction with oxytocin stimulation. These devices are typicallyprovided as a specialized breast pump which can be applied to the breastcovering the nipple, and which typically directly receives the expressedmammary fluid. In preferred methods involving use of a breast pump,negative pressure is generated on the breast to induce expression ofmammary fluid, optionally facilitated by prior or concurrentadministration of oxytocin. In yet additional alternative methods,mammary fluid can be expressed and collected without the aid of a breastpump, which may require an increase of oxytocin dosage or lengthening ofthe post administration time period before the mammary fluid is fullyexpressed from the nipple.

[0022] During or after mammary fluid expression, a biological sample iscollected from the expressed mammary fluid, which sample may consist ofwhole mammary fluid, whole cells, cell fragments, cell membranes,selected liquid, cellular or other solid fractions of the mammary fluid,as well as proteins, glycoproteins, peptides, nucleotides (including DNAand RNA polynucleotides) and other like biochemical and molecularconstituents of the mammary fluid.

[0023] Sample collection can be achieved simply by receiving theexpressed mammary fluid within any suitable reservoir, such as anordinary sample storage container or assay vessel. In preferredembodiments of the invention, the expressed mammary fluid is exposed toa solid phase sample collection medium, simultaneous with or subsequentto the time of breast fluid expression. Suitable solid phase media inthis context include microscopic glass slides, capillary tubes, coatedtubes, microtiter wells or plates, membranes, filters, affinity columns,dot blot matrices, beads, microspheres, resins, and other like mediathat will selectively adsorb, bind, filter, partition or otherwiseprocess desired components of the mammary fluid for convenientincorporation into a desired assay. Often it will be desirable tocombine a plurality of solid phase media for sample collection, e.g., afilter and membrane, a membrane and a particulate medium, etc., forexample to differentially partition and adsorb selected components ofthe breast fluid.

[0024] In conjunction with sample collection, the sample may be exposedto other agents such as buffers, diluents, extraction or chromatographicmedia, cross-linking agents, denaturing agents, etc., to stabilize orotherwise prepare the sample for processing within a desired assay.

[0025] Thus provided within the invention are methods and devices forobtaining a biological sample from a patient and/or determining theamount of a breast disease marker in a biological sample from breastfluid, which employ a novel breast pump or breast pump adapter. Thebreast pump functions in a similar fashion as a conventional breast pumpbut also provides a solid phase sample collection medium in fluidconnection with the pump. The solid phase sample collection medium maybe integrated within the breast pump or otherwise fluidly connectedtherewith, so that a sample of expressed mammary fluid contacts thecollection medium, typically while the pump remains applied to thebreast. In more detailed aspects of the invention, methods for employingthe novel breast pump include a step of applying the breast pump tostimulate breast fluid expression, with or without prior oxytocin orcarbetocin induction, wherein the solid phase sample collection mediumis fluidly connected with a breast engaging portion or member of thebreast pump.

[0026] According to these methods, operation of the pump results in anexpressed breast fluid sample contacting the solid phase samplecollection medium, typically while the pump remains applied to thebreast. Within the foregoing methods, additional methods are providedwhich employ a novel, hand-held breast pump device, wherein a doctor,technician or patient collecting a breast fluid specimen can grasp andoperate the hand-held pump to stimulate expression of breast fluid andcollect a specimen thereof while keeping one hand free for additionaltasks. The compact hand-held pump design allows the device to be pickedup and manipulated with one hand, to seat the breast engaging elementagainst the breast, apply vacuum pressure to the breast by manualoperation of the vacuum pump to cause a suitable volume of breast fluidto be expressed at or near the nipple, and to simultaneously collect atleast a primary sample of expressed breast fluid onto, or within, thesolid phase sample collection medium without additional manual steps ora need to remove the device from the breast or engage two hands in theoperation.

[0027] In certain collection methods of the invention, breast fluidexpressed by use of the general purpose or hand-held breast pump issimultaneously or subsequently diluted, filtered, washed, admixed withfixative or other processing agents, or otherwise processed or modifiedto yield a collected fluid sample partially or completely devoid ofcells, proteins and/or other selected components originally present inthe expressed fluid, to provide a processed fluid sample for laboratoryanalysis. In other embodiments, particulate components of the breastfluid, for example, cells, cellular components and/or cellular debris,are collected after processing and/or modification, e.g., forcytological examination. Often, primary sample collection and/orprocessing in this context is coincident with the fluid contacting oneor more solid phase collection medium(a) fluidly connected with thebreast engaging member. Depending on the type(s) of medium(a) used,preliminary sample processing can also be achieved directly by simpleoperation of the hand-held pump, without the need for additionalprocessing steps or removal of the breast engaging member from thesubject's breast.

[0028] In other alternative methods within the invention, preliminarysample processing involves additional steps following breast fluidexpression. In certain embodiments, the breast engaging member isremoved from the breast after the breast fluid is expressed and thefluid is transferred to a first solid phase sample collection medium,typically a membrane or filter. This initial or primary stage of samplecollection may be followed by washing or by manual transfer of selectedbreast fluid components (e.g., proteins, carbohydrates, cells, orcellular debris) from the first solid phase collection medium (e.g., anitrocellulose membrane) to a second solid phase medium, e.g., afluid-containing reservoir. Typically, preliminary sample processing inthis regard precedes final packaging of the collected sample for storageor shipment to a lab for further processing and analysis of the sample.In one example, whole cells or other cellular materials are separatedfrom expressed mammary fluid onto a nitrocellulose membrane or a filter,which is typically secured in fluid connection with the breast engagingmember by a fixed or removable support member mounted to the engagingmember or sample collection housing. The cells are subsequentlytransferred or washed in fluid (e.g., cytology fluid) to a second solidphase sample collection medium, for example a removable fluid reservoirconnected to, or integrated with, the breast engaging member or samplecollection housing.

[0029] In relation with these methods, various sample collection devicesand accessories for use therewith are provided within more detailedembodiments of the invention. Typically, breast fluid collection devicesof the invention include a breast engaging member constructed of anon-porous material that is sized and dimensioned to receive at least anipple portion of a human breast and form a suction seal therewith. Oneor more solid phase sample collection media are provided in fluidconnection with the breast engaging member for receiving a sample ofexpressed breast fluid. A vacuum pump mechanism is provided in gaseousconnection with the breast engaging member for generating negativepressure through the breast engaging member to facilitate breast fluidexpression.

[0030] In specific embodiments of the collection device of theinvention, a sample collection housing is fluidly connected with thebreast engaging member. The solid phase sample collection medium isoften removably supported within the housing in proximity to the nipplewhen the breast engaging member is applied to the breast and negativepressure is generated by the vacuum pump mechanism. The solid phasesample collection medium can be one or more microscopic glass slides,capillary tubes, collection tubes, vials, columns, micro-columns, wells,plates, membranes, filters, resins, inorganic matrices, beads, resins,particulate chromatographic media, plastic microparticles, latexparticles, coated tubes, coated templates, coated beads, or coatedmatrices.

[0031] Optional features of the breast fluid collection device includeremovable coupling means for removably coupling said sample collectionhousing with said breast engaging member. In other embodiments, thesolid phase sample collection medium may be supported by a supportmember integrally or removably mounted within the sample collectionhousing in fluid connection with said breast engaging member. Varioustypes of support members, including disposable or reuseable discs,cartridges and cassettes are provided as an accessory for use within theinvention. In yet additional embodiments, a reciprocating mechanism forreciprocally adjusting a position of the solid phase sample collectionmedium relative to the breast engaging member is incorporated within thedevice. The reciprocating mechanism may incorporate a support member orcarrier reciprocatingly mounted relative to the breast engaging member,which support member or carrier supports the solid phase samplecollection medium. Yet another optional feature of the device includes abreast pump adapter employing concepts of the invention for collectionof mammary fluid samples and operable in combination with a conventionalbreast pump.

[0032] In other detailed embodiments of the invention, the samplecollection device is a hand-held breast pump incorporating the breastengaging member and the vacuum pump mechanism in a compact, structurallyintegrated breast fluid collection apparatus suitable for manipulationand operation using only one hand. In certain embodiments, handheldbreast pump comprises a modular device made up of a plurality ofcomponents, each joined or securable in fixed structural interconnectionwith one another and capable of partial or complete disassembly fromremaining components to facilitate operation, cleaning, servicing and/orstorage of the device. The modular breast pump can include, for example,a separate breast engaging member detachable from one or moreinterconnecting components of the device for cleaning or to allowinterchanging of different engaging members to accommodate breastanatomy differences among patients.

[0033] Within more detailed embodiments of the hand-held breast pump,the solid phase sample collection medium can be supported by a supportmember removably mounted in fluid connection with the breast engagingmember. The support member can be a removable cassette for removableplacement in fluid connection with the breast engaging member. Thesupport member can house any of the above identified collection media,and may incorporate one or more air channels that pass through a body ofthe support member for passage of vacuum pressure and/or samplematerials between the breast engaging member and a sample collectionhousing member of the hand-held breast pump.

[0034] Within other detailed embodiments, the hand-held device includesa fluid-retaining recess, well or reservoir integrated or fluidlyconnected with the support member or a sample collection housing memberof the device. The fluid-retaining recess, well or reservoir maycomprise an integral, defined compartment or enclosure within the samplecollection housing for receiving the breast fluid and/or constituentsamples thereof. Alternatively, fluid-retaining recess, well orreservoir comprises a removable fluid reservoir member of the samplecollection housing, typically provided as a rigid sample collection tubeor vial removably connected with an outer casing member of the housing.The removable reservoir member is optionally sealably connected with theouter casing member of the housing to form an airtight couplingtherewith. In certain embodiments, the removable reservoir memberfeatures a circumferential ridge, fin, O-ring or other sealableengagement means to engage and make an airtight seal against a wall orother surface of the casing member when the vial is nested within thecasing member.

[0035] In additional detailed embodiments, the removable reservoirmember is gaseously and fluidly connected with the breast engagingmember to facilitate sample collection. For example, the vacuum pressurefrom the vacuum pump means may be routed to the breast engaging memberthrough the removable reservoir member of the housing, which is modifiedto include one or more air ports that form a gaseous connection betweena lumen of the reservoir and the vacuum pump means. The reservoir membermay function in this context as both a conduit for vacuum pressuretransmission to the breast and a receptacle for fluid sample materialsto directly collect expressed fluid or as a secondary collection mediumto receive primarily collected sample materials washed or otherwisetransferred from a primary solid phase sample collection medium into thereservoir. For example, a primary solid phase sample collection mediumfluidly connected with the breast engaging member may be positioned tocollect a primary sample of one or more breast fluid components whichcan thereafter be washed or otherwise transferred directly or indirectlyinto the removable reservoir member, without removal or disassembly ofthe breast engaging member and reservoir member.

[0036] The fluid collection reservoir may serve a dual purpose forcollection, as well as for storage, transport and/or processing ofcollected breast fluid or breast fluid component samples. Relating tothis purpose, the removable reservoir member further comprises closuremeans for closing the reservoir after sample collection is completed toprevent sample contamination and spillage. The closure means maycomprises a cap adapted to sealably engage a top end of the removablereservoir member. Where the reservoir member is modified to include oneor more air ports for transmission of vacuum pressure between the lumenof the reservoir and the vacuum pump means, the closure means includesecondary closure means to sealably close the air port(s) after samplecollection. For example, the secondary closure means may comprise anadhesive seal or sticker sized and constructed to adhere to an outerwall of the reservoir member surrounding an air port opening. Typically,the secondary closure means comprises a combined closure and labelingdevice which functions as a secondary closure mechanism to seal the airport(s) of the removable reservoir, and as a labeling template toprovide a writing surface for sample labeling. The combined closure andlabeling tab or sticker generally includes a first, closure-formingsurface for application over the air port to form a seal byjuxtaposition or adhesive contact with an outer wall of the removablereservoir, and a second, labeling surface opposite the closure-formingsurface made of a blank template material suitable for receiving astable, ink or graphite imprint. In more detailed aspects, the secondaryclosure means comprises a combined closure and labeling tab or stickerwhich is pre-attached to the removable reservoir member in a first, openconfiguration and which can be manually repositioned or otherwisemanipulated after sample collection to a second, closed configuration toform a seal or closure against the air port(s).

[0037] In related aspects of the invention, a novel breast fluidcollection reservoir, e.g., a modified cytology vial, is provided foruse within a mammary fluid collection device of the invention, whichreservoir incorporates the foregoing features of the removable reservoirmember of the sample collection housing. The novel collection reservoirthus provided is useful within the breast fluid collection methods ofthe invention, as well as within sample processing and diagnostic assaymethods performed in the laboratory subsequent to collection of a breastfluid sample.

[0038] In related aspects of the invention, methods are provided fordetermining the presence or amount of a breast disease marker,preferably a breast cancer marker, in biological samples obtained from amammary organ of a mammalian patient. These methods may involveintranasal, intramuscular or intravascular administration of oxytocin oran oxytocin analog to mammalian patients in amounts effective tostimulate mammary fluid expression in the patient. Once a sufficientpost-administration time period has elapsed to allow the oxytocin toreach and stimulate target alveolar-ductal tissues, mammary fluid iscollected directly from the nipple or, alternatively, the breast ispumped, and a biological sample from expressed mammary fluid iscollected, as above. After the sample is collected a bioassay isconducted on the sample to determine the presence and/or amount of thebreast disease marker in the sample. Suitable bioassays in this regardinclude assays to detect known markers of breast disease, such as assaysemploying immunological or other suitable probes to detect specificantigens and other markers expressed by selected pathogens, includingbacterial and viral pathogens. More preferred bioassays will detectindividual markers or panels of markers of benign breast tumors,pre-cancerous breast disease, and/or breast cancer, such as assaysemploying immunological or other suitable probes to detect specificantigens and other markers expressed by benign, pre-cancerous and/orcancerous alveolar-ductal cells of the breast. Preferably, the assaywill detect the presence or amount of multiple breast disease markers inthe biological sample, for example by including a panel of immunologicalor molecular probe(s) that bind or react with multiple breast cancermarkers.

[0039] Within further related aspects of the invention, novel methodsare provided handling or processing a biological sample of mammaryfluid, or a component thereof, for use in a diagnostic assay to detector quantify a breast disease marker in a biological sample of mammaryfluid. The methods typically include providing or obtaining thebiological sample of mammary fluid, or component thereof, in afluid-retaining reservoir that features a top end defining a primaryopening for access to the sample and an outer reservoir wall definingone or more air ports that communicate between the outer wall and aninner lumen of the vial. In certain aspects, the fluid-containingreservoir member includes closure means for closure of the reservoirafter the sample is introduced therein, to prevent sample contaminationand spillage.

[0040] The closure means may comprise a cap adapted to sealably engage atop end of the removable reservoir member. In various embodiments, theclosure includes secondary closure means to sealably close the airport(s) after the biological sample is introduced into said reservoir.Exemplary secondary closure means include an adhesive seal or stickerthat is sized and constructed to adhere to an outer wall of thereservoir member surrounding an air port opening. Alternatively, thesecondary closure means may include a combined closure and labelingdevice which functions as a secondary closure mechanism to seal the airport(s) of the removable reservoir and as a labeling template to providea writing surface for sample labeling. The combined closure and labelingtab or sticker may be directly applied to seal the air port after samplecollection having a first, closure-forming surface for application overthe air port to form a seal by juxtaposition or adhesive contact with anouter wall of the removable reservoir, and a second, labeling surfaceopposite the closure-forming surface made of a blank template materialsuitable for receiving a stable, ink or graphite imprint thereon.

[0041] Within additional aspects of the invention, the first,closure-forming surface of the reservoir can be provided with anadhesive coating resistant to disruption by contact with aqueoussolutions. The secondary closure means may include a combined closureand labeling tab or sticker which is pre-attached to the removablereservoir member in a first, open configuration and which can bemanually repositioned or otherwise manipulated after sample collectionto a second, closed configuration to form a seal or closure against theair port(s). The secondary closure means can optionally comprise anadhesive tab or strip folded in the open configuration to form an innerlayer affixed to the reservoir proximate to the air port, and an outerlayer folded over the inner layer, said outer layer providing the first,closure-forming surface and the second, labeling surface, wherein theouter layer can be unfolded away from the inner layer and wrapped aroundthe reservoir so that the closure-forming surface covers the air port toform a fluid-resistant closure and the labeling surface faces outwardfor recordation of sample data. The outer layer can be optionallysecured in a folded-back position against the inner layer by adhesiveengagement of the labeling surface with the inner layer.

[0042] In related embodiments, the first, closure-forming surface may beprovided with an adhesive coating that is protected in the openconfiguration by folding of an end segment of the outer layer bearingthe adhesive coating back, so that the closure forming surface providesa protective surface to shield the adhesive prior to closure, wherebythe end segment can be lifted and pulled outward to unfold the endsegment to separate the adhesive coating on the closure-forming surfacefrom the protective surface and to release the outer layer from theinner layer for closing of the air port(s).

[0043] In certain aspects of the invention, the foregoing methodsutilize a fluid-retaining reservoir that is a modified cytology vial. Inother aspects, the fluid-retaining reservoir comprises a removable fluidreservoir member of a sample collection housing of a mammary fluidcollection device. The fluid-retaining reservoir may be a rigid samplecollection tube or vial that is optionally removably connected with anouter casing member of a sample collection housing of said collectiondevice. The fluid-retaining reservoir can alternately be adapted forremovable, sealable connection with the outer casing member of thehousing to form an airtight coupling therewith. In exemplaryembodiments, the fluid-retaining reservoir is a cytology vial that issealably connectable with said outer casing member to form an airtightcoupling therewith. The fluid-retaining reservoir can optionally beremovably nested within the casing member to form a substantiallyairtight contact between an inner wall of the casing member wall and anouter wall, or a top or bottom end, of the reservoir member. In certainconfigurations, an outer wall of the fluid-retaining reservoir bears acircumferential ridge, fin or O-ring. The fluid-retaining reservoir canoptionally comprise a removable fluid reservoir member of the samplecollection housing of a mammary fluid collection device, and theabove-described circumferential ridge, fin or O-ring may be adapted toengage and make a circumferential airtight seal against an inner wall ofa casing member of the sample collection housing of the device.

[0044] In exemplary sample handling and processing methods of theinvention, the mammary fluid sample or component thereof includes wholecells or cell fragments. The handling and processing methods can furtherinclude the step of accessing the sample within reservoir to transfer orprocess the sample for detection or quantification of the breast diseasemarker. In exemplary embodiments, the sample may be transferred to asecond reservoir or other sample container or template for processingthe sample to detect or quantify the breast disease marker. Exemplarysteps of sample processing include detection or quantification of thebreast disease marker by staining cells or cell fragments from thesample with a cytological stain to detect a cytological marker.Alternative processing steps include detection or quantification of thebreast disease marker by microscopic examination of stained cells orcell fragments from the sample. Within certain embodiments, thebiological sample includes one or more components selected from thegroup consisting of whole mammary fluid, whole cells, cell fragments,cell membranes, purified proteins, bulk proteins, glycoproteins,peptides and polynucleotide components of a primary mammary fluidsample. Often, the breast disease marker will comprise a breast cancermarker. In various aspects, the breast disease marker is selected fromthe group consisting of a protein, a peptide, a glycoprotein, a lipid, aDNA polynucleotide and an RNA polynucleotide. Exemplary breast diseasemarkers include Ki67 Growth Factor, Cyclin D1, Proliferating CellNuclear Antigen, Transforming Growth Factor, Tissue PlasminogenActivator, Insulin Growth Factor Receptors, Collagenase Type IV, LamininReceptor, Integrins, p53, rb, nm23, ras, c-myc, Heat Shock Proteins,Prolactin, Neuron-Specific Enolase, IR-14, KA 1, KA 14,Alpha-Lactalbumin, Actin, CEA, HMFG, MCA, vasopressin and cathepsin.

[0045] Within the foregoing methods the breast disease markers may bedetected using any of a variety of useful techniques, for example byELISA immunoassay, immunoprecipitation assay, or solid phaseimmunoassay.

[0046] In yet additional aspects of the invention, clinically usefulkits are provided for determining the presence and/or amount of a breastdisease marker, preferably a breast cancer marker, in biological samplesobtained from a mammary organ of a mammalian patient. The kits include amammary fluid collection device in the form of a general purpose orhand-held breast pump as described herein. Additional kits include oneor more breast pump attachments (e.g., a detachable breast engagingmember, or multiple such attachments for use with different patients),accessories (e.g., replaceable fluid-retaining reservoirs), solid phasemedia, and/or disposable or reusable support members, cartridges orcassettes for holding collection media, as described herein. These andother kit components may be provided, alone or in any combination, withor without inclusion of the basic breast pump apparatus in the kit. Yetadditional kits include a pharmaceutical preparation of oxytocin or anoxytocin analog in a biologically suitable carrier for use in alternatemammary fluid collection methods of the invention. Still other kitsinclude on or more preparative and/or diagnostic reagents selected fromthose disclosed herein, including one or more fixatives, probes, labelsand the like in separate or common containers. In certain embodiments ofthe invention, kits include compositions and/or devices for detectingthe presence or amount of one or more breast disease marker(s) in thebiological sample, often including one or more immunological ormolecular probe(s) that binds or reacts with one or more breast cancermarker(s). The foregoing kit components are generally assembled in acollective packaging unit, which may include written or otherwiseuser-accessible instructions detailing the sample collection, handlingand/or processing methods of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0047]FIG. 1 is a partial sectional view of a breast pump employing theconcepts of the invention.

[0048]FIG. 2 is a sectional view of a portion of the breast pump asindicated in FIG. 1.

[0049]FIG. 3 is a perspective view of a support member for supporting asolid phase sample collection medium in fluid connection with a breastpump.

[0050]FIG. 4 is a perspective view of an alternative support member forsupporting a solid phase sample collection medium in fluid connectionwith a breast pump.

[0051]FIG. 5 is a perspective view of an alternative support member forsupporting a solid phase sample collection medium in fluid connectionwith a breast pump.

[0052]FIG. 6 is a perspective view of an alternative support member forsupporting a solid phase sample collection medium in fluid connectionwith a breast pump.

[0053]FIG. 7 is a perspective view of an alternative support member forsupporting a solid phase sample collection medium in fluid connectionwith a breast pump.

[0054]FIG. 8 is a partial sectional view of a breast pump deviceemploying the concepts of the invention.

[0055]FIG. 9 is a partial sectional view of a portion of a breast pumpillustrating a support member and cartridge for containing a particulatesolid phase sample collection medium.

[0056]FIG. 10 is a partial sectional view of a portion of a breast pumpillustrating a support member and an exemplary solid phase samplecollection template (coated tube).

[0057]FIG. 11 is a partial sectional view of a breast pump employing areciprocating mechanism to adjust positioning of a solid phase samplecollection medium within the pump.

[0058]FIG. 12 is a sectional view depicting a breast pump adapteremploying the concepts of the invention.

[0059]FIG. 13 is a sectional view depicting a breast pump adapteremploying a reciprocating mechanism to adjust positioning of a solidphase sample collection medium within the adapter.

[0060]FIGS. 14 and 15 provide partial sectional views of a breast pumpemploying a sliding reciprocating mechanism to adjust positioning of asolid phase sample collection medium within the pump.

[0061]FIG. 16 is a sectional view illustrating a hand-held breast pumpof the invention.

[0062]FIG. 17 is a sectional view illustrating a hand-held breast pumpof the invention.

[0063]FIG. 18 is an exploded perspective view illustrating a hand-heldbreast pump of the invention employing a removable support member andremovable fluid reservoir for primary and secondary sample collection.

[0064]FIGS. 19 and 20 provide perspective views of alternativeembodiments of a removable sample collection reservoir for use with thehand-held breast pump of the invention.

[0065] FIGS. 21-23 provide sectional views of the removable fluidreservoir of FIG. 19, illustrating operation of a closure/labeling stripto seal the reservoir and provide an exterior labeling surface forrecordation of sample data.

[0066] FIGS. 24-26 provide top plan views of a modified membrane orfilter for use within the breast pump of the invention havingperforations or slits for enhancing permeability and flexibility of themembrane or filter.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0067] As noted above, the invention provides methods and devices 10,10′ for obtaining, handling, and processing biological samples frommammary fluid. Preferably, these methods are non-invasive, meaning theyare non-surgical and do not involve penetration of the breast by needlesor other intrusive devices. To practice the non-invasive samplecollecting method, the invention provides specialized breast pumpdevices which feature a breast engaging portion or member coupled with avacuum pump mechanism and fluidly connected with a solid phase samplecollection medium. The mammary fluid collection devices and methodsdisclosed herein are related in certain aspects to mammary fluidcollection devices and methods disclosed in U.S. patent application Ser.No. 09/435,131, filed Nov. 5, 1999, U.S. patent application Ser. No.09/027,362, filed Feb. 20, 1998, and U.S. Pat. No. 5,798,266, issuedAug. 25, 1998, each incorporated herein by reference. The devices andmethods of the present invention are effective to induce mammary fluidexpression for sample collection, alone or in conjunction with oxytocinstimulation.

[0068] The mammary fluid collection devices of the invention aretypically provided as a specialized breast pump 10, 10′ that can beapplied to a human or animal breast covering the nipple, and whichtypically receives expressed mammary fluid within a solid phase samplecollection medium, and in some cases within a removable samplecollection reservoir. During or after mammary fluid expression, abiological sample is collected from the expressed mammary fluid, whichsample may consist of whole mammary fluid, whole cells, cell fragments,cell membranes, selected liquid, cellular or other solid fractions ofthe mammary fluid, as well as proteins, glycoproteins, peptides,nucleotides (including DNA and RNA polynucleotides) and other likebiochemical and molecular constituents of the mammary fluid.

[0069] The breast pump devices 10, 10′ of the invention function in partby generating negative pressure applied to the nipple area of the breastto induce mammary fluid expression. Fluid expression induced by thesebreast pump devices may optionally be facilitated by coordinateadministration of the peptide hormone oxytocin, or a functional analogthereof, in an amount effective to stimulate or increase expression ofmammary fluid induced by the breast pump device. The oxytocin oroxytocin analog (for example a longacting oxytocin analog such ascarbetocin) is administered in a manner (e.g., intranasally) and amountsufficient to reach and stimulate a target alveolar-ductal tissue of thebreast, whereby the oxytocin stimulates myoepithelial contraction of thealveolar-ductal tissue to induce or facilitate mammary fluid expression.

[0070] During or after the mammary fluid expression step, a biologicalsample is collected from the expressed mammary fluid. A range ofsuitable biological samples are contemplated and will be useful withinthe methods of the invention, including whole mammary fluid, selectedliquid or solid fractions of the mammary fluid, whole cells or cellularconstituents, proteins, glycoproteins, peptides, nucleotides (includingDNA and RNA polynucleotides) and other like biochemical and molecularconstituents of the mammary fluid. Sample collection can be achievedsimply by receiving the expressed mammary fluid within a suitablereservoir, such as an ordinary sample storage container or assay vessel.

[0071] In preferred embodiments of the invention, the expressed mammaryfluid is contacted with a solid phase sample collection medium fluidlyconnected with the breast pump 10, 10′, simultaneous with or subsequentto the time of breast fluid expression. Suitable solid phase media inthis context include microscopic glass slides, capillary tubes, coatedtubes, microtiter wells or plates, membranes, filters, affinity columns,dot blot matrices, beads, resins, and other like media that willselectively adsorb, bind, filter, partition or otherwise process desiredcomponents of the mammary fluid for convenient incorporation into adesired assay.

[0072] A wide range of sample collection procedures and materials knownin the art are useful within the invention. Selected methods andmaterials will vary among different assays, as will be understood andreadily practiced by those skilled in the art. For example, if thebreast disease marker sought in a particular assay is a soluble protein,it will often be desired to immobilize the protein on a solid phasematrix or template by contacting the target protein with a reagenthaving high specificity for the protein, preferably a polyclonal ormonoclonal antibody. The yields a complex, e.g., a ligand-proteincomplex, an antibody-antigen complex, or other complex in which thetarget protein is bound to a specific binding partner (i.e., wherein thecomplex is not dissociated upon addition of a non-specific bindingpartner conventionally used as a control to determine specific binding;and preferably wherein the binding partner binds with an affinity of kD10-9 or greater). The binding partner that binds to the target proteinis in turn immobilized to the solid phase medium, before or aftercomplex formation with the target protein. Immobilization of the bindingpartner, e.g., by covalent binding to a solid phase template or matrix,can be achieved by a variety of conventional methods known in the art.

[0073] In this manner, the target protein/binding partner complex isadsorbed or otherwise bound directly to an insoluble matrix.Alternatively, a variety of secondary binding partners, e.g.,anti-isotype antibodies, may be added to bind the complex to theinsoluble matrix. The latter step depends on the nature of the firstbinding partner (i.e., the binding agent that specifically binds thetarget protein), for example whether the first binding partner is aprimary antibody, ligand, etc.

[0074] Particularly useful within the invention are immunoassay whichformats employ a combination of solid phase or immobilized reagents andlabeled reagents whereby the association of the label with the solidphase is a function of the presence or absence of reactivity with thetargeted antigen. In general, such a solid phase reagent comprises abinding substance such as an anti-antibody (e.g., anti-IgG), or otherimmunobinder or other binding agent according to the assay protocolinvolved, bound or attached, covalently or noncovalently, to the solidphase matrix or in an otherwise immobilized form.

[0075] Useful labeled reagents in solid phase immunoassays include abinding substance such as an anti-antibody (e.g., anti-IgG), or otherimmunobinder or other binding agent according to the assay protocolinvolved, which is chemically coupled with a detectable chemical moiety.Useful labels are conventional in the art and include fluorescers,chemiluminescers, radioisotopes, and enzymes. Enzyme labels areparticularly useful and are generally selected from alkalinephosphatase, peroxidase, and P-galactosidase. Enzyme labels are readilydetectable by addition of a corresponding chromogenic substrate anddetecting the resulting color or fluorescent response.

[0076] A variation of this protocol uses a ligand-modified form of thetargeted antigen(s) with immobilization to the solid phase beingaccomplished by using a solid phase bearing an immobilized (e.g., boundor adsorbed) binding partner to the ligand. For example, biotin or ahapten (e.g., fluorescein) can be used as the ligand and can beimmobilized by contact with a solid phase form of avidin or anti-haptenantibody, respectively. The addition of the solid phase binding partnercan occur at any convenient time in the assay, such as prior to contactof sample with the ligand-antigens(s) or thereafter.

[0077] Preferred solid phase matrices for use within the foregoingmethods include Staphylococcus aureus or Protein A or G Agarose [e.g.Sepharose® (Pharmacia Biotech AB, Uppsala, Sweden)] beads. Protein A andprotein G are cell wall proteins isolated from specific bacterialstrains, and have specific binding sites for certain classes ofimmunoglobulins. Protein A binds (to varying degrees) most subclasses ofIgG, plus IgM, IgA, and IgD. Protein G binds nearly all subclasses ofIgG, but not other classes of immunoglobulins.

[0078] An alternative solid phase sample collection and/or assay methodutilizes a specific anti-marker primary antibody that is covalentlyattached to the solid phasematrix, e.g., by covalent linking theantibody through its free amino groups to cyanogen-bromide activatedSepharose particles. Insolubilized antibody can be used to pull thecorresponding marker antigen out of solution by adsorption to itssurface. In yet another alternative format, the marker protein can betreated with a cross-linking reagent (e.g. biotin or digoxigenin) thatmay be subsequently detected by a second binding partner. In the case ofbiotin, the second binding partner is avidin or streptavidin; fordigoxigenin, the second reagent is an anti digoxigenin antibody. Avidinand streptavidin may be coupled directly to the solid phase medium,e.g., to agarose beads. Because the initial biotinylation is notspecific for the marker, samples are frequently electrophoresed on,e.g., SDS PAGE, transferred to nitrocellulose etc., and Western blottedwith antibodies specific for the protein factor.

[0079] A preferred assay method for detecting protein markers is thewell known, Enzyme Linked Immunosorbant Assay (ELISA) assay. Accordingto this method, a variety of coating reagents can be adsorbed orotherwise bound directly onto a surface of a desired solid phase samplecollection medium, e.g., a microtiter plate, well, tube, bead, teststrip, plastic microparticle, latex particle, etc., to form a coatedtemplate or matrix. These coating reagents are typically aspecies-specific anti-isotype antibody (e.g., anti-mouse-IgG) but canalso include an anti-marker primary antibody or an affinity reagent suchas avidin or streptavidin. The target protein (e.g., a soluble proteinmarker) is contacted with a specific primary antibody or, alternatively,is crosslinked (e.g., to biotin) or otherwise modified to form acomplex, and the resulting complex is adsorbed to the coated template ormatrix and processed according to conventional assay methods.

[0080] Latex or particle agglutination methods are also to be mentioned.Particles are coated or covalently coupled with a target antigen,ligand, antibody or other binding partner. The particles are thenincubated with a test sample and resulting agglutination of theparticles, e.g., due to formation of ICA antibody linkages betweenparticles, is detected. Detection can be accomplished by visualobservation (e.g., a slide agglutination format) or quantified bymeasuring turbidity changes with a spectrophotometer or nephelometer. Awell known variation of this general method based on inhibition ofparticle agglutination can also be employed. In addition, anagglutinator reagent can be prepared comprising multiple antigens, e.g.,a water soluble polymer backbone to which are attached multiples of oneor more antigens within a panel.

[0081] Alternative methods for collecting and analyzing samples withinthe invention include Western immunoblot and dot-blot methods. Forapplication of these methods, the solid phase sample collection mediumis preferably a membrane or filter, e.g., a nitrocellulose,polyvinylidene difluoride (PVDF), or nylon membrane. Proteins within thebreast fluid sample may be processed (e.g., separated on SDS PAGE) ordirectly transferred to the membrane, and non-specific interactions maybe blocked by incubating the membrane with, e.g., bovine serumalbumin/ovalbumin or non-fat dry milk. A primary antibody withspecificity for the protein marker is contacted with the membrane, andexcess antibody is washed, e.g., with buffered detergent. A labeledisotype specific antibody is next contacted with the membrane, andtarget protein-primary antibody-secondary antibody ternary complexes aredetected, e.g., calorimetrically.

[0082] Where the targeted protein factor includes a carbohydrate moiety,the factor can also be adsorbed to a solid phase template or matrix,e.g, a resin, by way of lectin-carbohydrate interactions. Variouslectins are available for this purpose that differ in their carbohydratebinding specificity. For example, Lectin Con A binds tomannose-containing carbohydrate structures and with low affinity toα-glucose and α-N-acetylglucosamine. Lectin GNA binds to terminalmannose residues. Lectin MAA binds to α(2-3) Linked sialic acids. Avariety of other lectins collectively providing a wide range ofspecificities are known in the art.

[0083] A particularly preferred solid phase sample collection medium foruse within the invention is a filter, pad or membrane that can bedirectly contacted to a sample of expressed breast fluid to adsorb,absorb, bind, partition or otherwise facilitate sample processing orhandling within a selected assay. For this purpose, several types oftransfer membranes are known, including nitrocellulose that is the mostcommonly used transfer membrane. Several commercial sources now offernitrocellulose impregnated with a synthetic support that improves itsdurability and flexibility without altering its performance. Onepreferred transfer membrane, polyvinylidene difluoride (PVDF), marketedby Millipore (Bedford, Mass.) under the trade name Immobilon®, hasslightly lower protein-binding capacity than nitrocellulose but ismechanically stronger and compatible with many organic solvents. Thisallows direct protein staining with Coomassie Blue, and direct aminoacid composition and sequence analysis of transferred proteins, withoutinterfering with its subsequent use for antibody probing.

[0084] Membranes are not only useful within the invention for proteinblotting, but also for immobilization of nucleic acids. Thus,nitrocellulose, reinforced nitrocellulose, diazotized membranes (paperor nylon), nylon, charged nylon, or PVDF, and DEAE-anion exchangemembranes are useful for immobilizing DNA and RNA from expressed breastfluid. In this context, the most commonly used membranes are reinforcednitrocellulose and nylon. Nitrocellulose has a lower background but alsoa lower binding capacity than nylon and is chosen primarily whenbackground, but not detectability, is the main concern. Nylon, incontrast, is ideal for lower copy number sequences, short targetsequences (down to oligomers) or for reprobing. Membranes are alsoavailable with different pore sizes. For DNA blots, membranes with apore size of 0.45 μm are usually chosen for large fragments, but 0.22 μmfor fragments of <500 bases. For RNA blots, membranes with a pore sizeof 0.1 or 0.22 μm are most efficient. Membranes are available indifferent size specifications, including sheets, rolls, pre-cut circles,etc.

[0085] Methods for detecting DNA on nylon without DNA purification andprocessing of the samples, e.g., for detecting DNA from fluids or wholecells, have recently been developed (Reed and Matthaei, Nucleic AcidsRes. 18:3093, 1990; and Hammermueller et al., J. Virol. Methods 31:47,1991; each incorporated herein by reference). These procedures avoidenzymatic dispersion of cells, RNase and pronase treatments to hydrolyzecellular macro-molecules, etc., and are typically based on the capacityof alkali and other reagents to disperse and solubilize cells andhydrolyze macro-molecules including RNA and protein, but not DNA.Positively charged modified nylon membranes then irreversibly bindnucleic acid while remaining suitable for hybridization.

[0086] Nucleic acid extraction and processing steps may also beminimized by well known fast blot methods. In particular, fast blotmethods that use nylon as a solid phase take advantage of the ability ofNaOH to dissociate cells, denature DNA and immobilize DNA.Nitrocellulose membranes have a lower binding capacity andco-immobilization of nucleic acid and protein from neutral solutions canbe a problem. Concentrated NaI can be used to inhibit proteinimmobilization, to denature DNA and to irreversibly bind the nucleicacid to nitrocellulose without a requirement for baking. This method canalso be used for RNA.

[0087] Although it is possible to directly transfer proteins, nucleicacids and other markers to a solid phase matrix which is in turndirectly incorporated in an assay, it may be desirable to concentratethe target marker, e.g., by chromatography, extraction, specific ornonspecific adsorption, etc., particularly when sensitivity is aproblem. Thus, samples can be collected and initially processed bycontacting breast fluid with a solid phase chromatographic medium, e.g.,within a cartridge comprising a micro-column of Sepharose-coupledantibody. Up to 500-fold increases in immunoassay sensitivity withapparent recoveries of 85 to 95% can be achieved using this approach.This and other well known chromatographic procedures provide a powerfulapproach to the quantitation of substances too dilute to be measured byroutine methods.

[0088] For sample collection and processing using chromatographic andrelated methods, a particulate solid phase sample collection medium ispreferred. Various particulate media are known which selectively adsorb,absorb, bind, or partition components of biological samples, which mediaare readily adapted for collection and processing of breast fluidsamples. These particulate can be coupled with various coating reagentsknown in the art, e.g., affinity reagents, to provided a coated medium,or may be used in an unmodified form.

[0089] Exemplary particulate sample collection media for use within theinvention include beads, plastic microparticles, latex microspheres,glass materials such as controlled porous glass, granular agarose basedmaterials, cross-linked dextran polymers, inorganic or organic ionexchanger materials, kieselsur and other silicate materials. Suitablematerials additionally include cellulosic materials, e.g.,diethylaminoethyl (DEAE) cellulose or diethylamino (DEA) cellulose. Alsouseful are natural polymeric carbohydrates and their syntheticallymodified, cross-linked or substituted derivatives, such as agar, agaroseand cross-linked dextran polymers.

[0090] Synthetic polymers which can be prepared with suitably porousstructures, such as vinyl polymers (e.g., polyethylene, polypropylene,polystyrene, polyvinylchloride, polyvinylacetate and its partiallyhydrolysed derivatives, polyacrylates, polyacrylamides,polymethacrylates), copolymers and terpolymers of the above vinylmonomers among themselves and with other monomers, polycondensates(e.g., polyesters and polyamides), and addition polymers, such aspolyurethanes or polyepoxides are also useful.

[0091] Yet additional particulate media are prepared from inorganicmaterials having a suitably porous form, such as sulfates or carbonatesof alkaline earth metals and magnesium. Examples include barium sulfate,calcium sulfate, calcium carbonate, magnesium carbonate, silicates ofalkali and alkaline earth metals and/or aluminum and/or magnesium, andaluminum or silicon oxides or hydrates, such as clays, alumina, talc,kaolin, zeolite, among others.

[0092] Also included among useful solid phase sample collection mediaporous barrier materials suitable for use with breast pump and breastpump adapter devices of the invention, for example to encloseparticulate solid phase media within a cartridge adapted for coupling influid connection with a breast pump or breast pump adapter. Such porousbarrier materials are inert to and nonreactive with markers and otheranalytes and reagents used in assaying for breast disease markers, andare porous with respect to the passage of liquids and/or particulates ofa pre-selected size. Suitable materials include various porous materialssuch as nylon fabric, polyethylene and other plastic films, membranes,filters, glass wool, sponge, styrofoam, ceramic and other porousmaterials.

[0093] In conjunction with sample collection, samples of expressedbreast fluid may be exposed to other agents such as buffers, diluents,extraction or chromatographic media, cross-linking agents, blockingagents, denaturing agents, etc., to stabilize or otherwise prepare thesample for processing within a desired assay. For example, the samplemay be diluted (e.g., by collecting the sample in a well or recesscontaining the solid phase medium wetted or suspended in a diluent) tominimize nonspecific binding effects, e.g, affecting a subsequentimmunoassay. In the exemplary context of sample collection forimmunoassays, the avidity of the antibody for the marker antigen is animportant consideration, whereby providing more or less diluent duringsample collection and incubation may optimize a particularantigen-antibody system being studied.

[0094] Commonly used buffers for dilution include phosphate, borate, orTris-buffered saline. Usually, the choice of the buffer is notimportant. Nonetheless, a careful examination of the effect of buffer,pH, ionic strength, and divalent cations will facilitate use of a newsample collection/assay system in order to maximize sensitivity andresolve possible sources of interference within the assay. Althoughimmunoassays are usually carried out at neutrality, doing so is notalways optimal.

[0095] Nonspecific binding or adsorption, e.g., of antigens and haptens(especially hydrophobic haptens) to glass and plastic tubes or pipettesmay markedly influence measured activity in a particular immunoassay.With some proteins and polypeptides, nonspecific binding in immunoassaysis reduced if plastic tubes are used. The addition of protein to themedium may also minimize nonspecific adsorption and help avoiddenaturation of highly diluted antigens and antibodies. Therefore,assays involving iodinated antigens are generally carried out inprotein-containing buffers. Bovine serum albumin, gelatin, lysozyme, andovalbumin are commonly used, usually at final concentrations of 1 to 5mg/ml. In some systems diluted whole serum or proteins present in thesample itself are just as satisfactory. However, even though addedproteins are often beneficial, they should not be used indiscriminatelywithout making an evaluation for possible adverse effects, for examplecontaminating enzymes that may degrade the marker protein.

[0096] Other possible additives for improved sample collection and assaymethods, apart from buffer and protein, include enzyme inhibitors andchelating agents. In assays lasting longer than 3 days, a bacteriostaticagent, such as sodium azide, 0.1 to 0.2%, may also be incorporated intothe sample collection and/or assay medium to help avoid microbialgrowth.

[0097] Although a fundamental utility of the present invention lies inthe novel, noninvasive methods for obtaining biological samples frommammary fluid, additional methods are disclosed herein that provideuseful assays for detecting and/or measuring important breast diseasemarkers in these samples. In this context, the invention provides abroad range of assay methods incorporating known procedures and reagentsfor determining the presence and/or expression levels of breast diseasemarkers, particularly breast cancer markers, in biological samples. Asincorporated within the invention, these methods involve application ofa breast pump 10, 10′ to mammalian patients, optionally coupled withoxytocin administration in amounts effective to facilitate mammary fluidexpression in the patient. After the sample is collected, a bioassay isconducted on the sample to determine the presence and/or amount of aselected breast disease marker, preferably a breast cancer marker orpanel of breast cancer markers, in the sample.

[0098] As used herein, the term breast disease marker refers to anycell, cell fragment, protein, peptide, glycoprotein, lipid, glycolipid,proteolipid, or other molecular or biological material that is uniquelyexpressed (e.g. as a cell surface or secreted protein) by diseasedbreast cells, or is expressed at a statistically significant, measurablyincreased or decreased level by diseased breast cells, or in associationwith breast disease (e.g. a protein expressed by an infectious agentassociated with breast disease), or is expressed at a statisticallysignificant, measurably increased or decreased level by diseased breastcells compared to normal breast cells, or which is expressed bynon-diseased breast cells in association with breast disease (e.g. inresponse to the presence of diseased breast cells or substances producedtherefrom). Breast disease markers can also include specific DNA or RNAsequences marking a deleterious genetic change, or an alteration inpatterns or levels of gene expression significantly associated withbreast disease. Preferred breast disease markers include markers ofbreast infections, benign neoplasia, malignant neoplasia, pre-cancerousconditions, and conditions associated with an increased risk of cancer.

[0099] As used herein, the term breast cancer marker refers to a subsetof breast disease markers, namely any protein, peptide, glycoprotein,lipid, glycolipid, proteolipid, or other molecular or biologicalmaterial that is uniquely expressed (e.g. as a cell surface or secretedprotein) by cancerous cells, or is expressed at a statisticallysignificant, measurably increased or decreased level by cancerous cellscompared to normal cells, or which is expressed by non-cancerous cellsin association with cancer (e.g. in response to the presence ofcancerous cells or substances produced therefrom). Breast cancer markerscan also include specific DNA or RNA sequences marking a deleteriousgenetic change, or an alteration in patterns or levels of geneexpression significantly associated with cancer. In addition, breastcancer markers can include cytological features of whole cells presentin mammary fluid, such as nuclear inclusions or cytoplasmic structuresor staining attributes uniquely expressed by, or associated with,cancerous cells.

[0100] Among the breast cancer markers that are useful within themethods of the invention, a subset are described in representativereview articles by Porter-Jordanet al., Hematol. Oncol. Clin. NorthAmer. 8:73-100, 1994; and Greiner, Pharmaceutical Tech, May, 1993, pp.28-44, each incorporated herein by reference in its entirety. Othersuitable markers are also widely known and can be readily incorporatedinto the methods of the invention using information and methodsgenerally known or available in the literature. Preferred breast cancermarkers for use within the invention include well characterized markersthat have been shown to have important value for determining prognosticand/or treatment-related variables in human female patients. As notedpreviously, prognostic variables are those variables that serve topredict outcome of disease, such as the likelihood or timing of relapseor survival. Treatment-related variables predict the likelihood ofsuccess or failure of a given therapeutic program. Determining thepresence or level of expression or activity of one or more of thesemarkers can aid in the differential diagnosis of patients with malignantand benign abnormalities, and can be useful for predicting the risk offuture relapse or the likelihood of response to a selected therapeuticoption.

[0101] It is important to note, however, that the invention does notrely solely on breast disease markers that meet the stringentrequirements of sensitivity and specificity that would render the markerimmediately acceptable for clinical application to human patients. Onthe contrary, a number of breast disease markers contemplated within theinvention fall short of these stringent criteria, and nonethelessprovide useful information that can be of substantial benefit indetecting, differentially diagnosing or managing breast cancer. Suchnon-clinically accepted markers are useful for immediate applicationwithin the methods of the invention as basic research tools, and asadjunctive tools in clinical applications. Beyond these immediateapplications, many such markers are expected to be further developed andrefined according to the methods of the invention to the point of directclinical applicability, particularly in assay methods that analyzecombinations of markers to generate complementary data of greaterpredictive value than data yielded by individual markers alone.

[0102] The preferred assay methods of the invention particularly focuson breast cancer markers associated with tumorigenesis, tumor growth,neovascularization and cancer invasion, and which by virtue of thisassociation provide important information concerning the risk, presence,status or future behavior of cancer in a patient. As noted previously,tumorigenesis and tumor growth can be assessed using a variety of cellprolferation markers (for example Ki67, cyclin D1 and PCNA). Tumorgrowth can also be evaluated using a variety of growth factor andhormone markers (for example estrogen, EGF, erbB-2, and TGF-α, receptorsof autocrine or exocrine growth factors and hormones (for example IGFand EGF receptors), or angiogenic factors. In addition to tumorigenic,proliferation and growth markers, a number of markers provideinformation concerning cancer invasion or metastatic potential in cancercells, for example by indicating changes in the expression or activityof cell adhesion or motility factors. Exemplary markers in this contextinclude Cathepsin D, plasminogen activators and collagenases. Inaddition, expression levels of several putative tumor “suppressor”genes, including nm23, p53 and rb, provide important data concerningmetastatic potential, or growth regulation of cancer cells. A largenumber and variety of suitable breast cancer markers in each of theseclasses have been identified, and many of these have been shown to haveimportant value for determining prognostic and/or treatment-relatedvariables relating to breast cancer.

[0103] Prior to or concurrent with each assay run of the invention, itmay be preferable to perform a preliminary evaluation to verify sampleorigin and/or quality. The focus of such preliminary evaluations is toverify that the sample collected from expressed mammary fluid is indeedof mammary origin, and is not contaminated with other potentialcontaminants, such as sweat from skin surrounding the nipple. For thesesample verification purposes, a variety of assays are available whichidentify mammary fluid markers known to be present in mammalian mammaryfluid, and which are preferably highly specific markers for mammaryfluid (i.e. markers which are typically always present in mammary fluidand which are absent from all, or most of, other potentiallycontaminating bodily fluids and tissues). However, an acceptable levelof specificity for mammary fluid markers within the methods of theinvention is provided by markers that are simply known to be present inmammary fluid, even though they may be present in other bodily fluids.One such marker is the enzyme lysozyme, which is a normal component ofhuman serum, urine, saliva, tears, nasal secretions, vaginal secretions,seminal fluid, and mammary fluid. Lysozyme (muramidase) is an enzymethat hydrolyzes beta 1,4-glycosidic linkages in the mucopolysaccharidecell wall of a variety of microorganisms resulting in cell lysis.Quantitative measurement of lysozyme is readily accomplished by a wellknown agar plate diffusion method, described in detail in theinstructions provided with the Quantiplate® lysozyme test kit, availablefrom Kallestad, Sanofi Diagnostics (Chasta, Minn.), incorporated hereinby reference in its entirety.

[0104] Other mammary fluid markers for sample verification that are morespecific than lysozyme are preferred within the methods of theinvention, and can be readily incorporated within the invention based onpublished and generally known information. The most preferred amongthese markers are proteins and other biological substances that arespecifically expressed or enriched in mammary fluid. A diverse array ofsuitable markers in this context have been characterized and havealready been used to develop specific antibodies, including affinitypurified and monoclonal antibodies. These antibodies can in turn beemployed as immunological probes to determine the presence or absence,and/or to quantify, selected mammary fluid markers to verify mammaryfluid sample origin and quality. Mammary fluid markers of particularinterest for use within the invention include specific cytokeratins thatare characteristically expressed by normal and cancerous mammaryepithelial cells, against which specific panels of antibody probes havealready been developed. (See for example, Nagle, J., Histochem.Cytochem. 34:869-881, 1986, incorporated herein by reference in itsentirety). Also useful as mammary fluid markers are the human mammaryepithelial antigens (HME-Ags) corresponding to glycoprotein componentsof the human milk fat globulin (HMFG) protein, against which specificantibodies (e.g., anti HMFG1, Unipath, U.K.) are also available. (SeeRosner et al, Cancer Invest. 13:573-582, 1995; Ceriani et al. Proc.Natl. Acad. Sci. USA 74:582-586, 1982; Ceriani et al., Breast CancerRes. Treat. 15:161-174, 1990, each incorporated herein by reference inits entirety).

[0105] To conduct the breast disease marker assays provided within theinvention, a collected biological sample from mammary fluid is generallyexposed to a probe that specifically binds to a selected breast diseaseor breast cancer marker, or otherwise interacts with the marker in adetectable manner to indicate the presence or absence, or amount, of thebreast disease or breast cancer marker in the sample. Selected probesfor this purpose will generally depend on the characteristics of thebreast disease marker, i.e. on whether the marker is a proteinpolynucleotide or other substance. In preferred embodiments of theinvention, the breast disease marker is a protein, peptide orglycoprotein, all of which are effectively targeted in breast diseasemarker assays using specific immunological probes. These immunologicalprobes can be labeled with a covalently bound label to provide a signalfor detecting the probe, or can be indirectly labeled, for example by alabeled secondary antibody that binds the immunological probe to providea detectable signal.

[0106] General methods for the production of non-human antisera ormonoclonal antibodies (e.g., murine, lagormorpha, porcine, equine) arewell known and may be accomplished by, for example, immunizing an animalwith a selected breast disease marker protein, peptides synthesized toinclude part of the marker protein sequence, degradation productsincluding part of the marker protein sequence, or fusion proteinsincluding all or part of the marker protein linked to a heterologousprotein or peptide. Within various embodiments, monoclonal antibodyproducing cells are obtained from immunized animals, immortalized andscreened, or screened first for the production of an antibody that bindsto the selected breast cancer marker protein or peptide, and thenimmortalized. It may be desirable to transfer the antigen bindingregions (i.e., F(ab′)2 or hypervariable regions) of non-human antibodiesinto the framework of a human antibody by recombinant DNA techniques toproduce a substantially human molecule. Methods for producing such“humanized” molecules are generally well known and described in, forexample, U.S. Pat. No. 4,816,397 (incorporated herein by reference inits entirety). Alternatively, a human monoclonal antibody or portionsthereof may be identified by first screening a human B-cell cDNA libraryfor DNA molecules that encode antibodies that specifically bind to theselected breast disease marker according to the method generally setforth by Huse et al. (Science 246:1275-1281, 1989 (incorporated hereinby reference in its entirety). The DNA molecule may then be cloned andamplified to obtain sequences that encode the antibody (or bindingdomain) of the desired specificity.

[0107] Also contemplated within the invention are bifunctionalantibodies having independent antigen binding sites on eachimmunoglobulin molecule (as disclosed for example in Thromb. Res. Suppl.X: 83, 1990, and in The Second Annual IBC International Conference onAntibody Engineering, A. George ed., Dec. 16-18, 1991; each incorporatedherein by reference in its entirety), as well as panels of individualantibodies having differing specificities. Bifunctional antibodies andantibody panels of particular use within the invention includeantibodies and panels of antibodies that bind to two or more selectedbreast disease markers to generate complementary data of greaterpredictive value than data yielded by individual markers alone.

[0108] Monoclonal antibodies are particularly useful within theinvention as labeled probes to detect, image and/or quantify thepresence or activity of selected breast disease markers. In thiscontext, monoclonal antibodies that specifically bind to selected breastdisease markers are provided which incorporate one or more well knownlabels, such as a dye, fluorescent tag or radiolabel. By incorporatingsuch a label, the antibodies can be employed in routine assays todetermine expression, localization and/or activity of one or moreselected breast disease markers in a biological sample including, orderived from, mammary fluid. Results of these assays to determineexpression, localization and/or activity of a selected breast diseasemarker in a test sample taken from a patient at risk for breast disease,or known to have breast disease, can be compared to results from controlstudies detecting and/or quantifying the same marker in biologicalsamples obtained from normal patients negative for breast disease. Inthis manner, baseline data and cutoff values can be determined accordingto routine methods to refine the assays of the invention and adapt themfor direct clinical application.

[0109] Detection and/or quantification of breast disease markers in thebiological samples of the invention can be accomplished using a varietyof methods. Preferred methods in this regard include well known ELISAimmunoassays, immunoprecipitation assays, and various solid phaseimmunoassays including Western blotting, dot blotting and affinitypurification immunoassays, among other methods. Comparable methods aredisclosed herein, or are elsewhere disclosed and known in the art, forusing non-antibody probes to detect and/or quantify the expressionand/or activity of breast disease markers. Suitable non-antibody probesfor use within the invention include, for example, labeled nucleotideprobes that hybridize at moderate or high stringency to DNA transcriptsof oncogenes and other DNA sequences associated with elevated breastdisease risk, or with mRNA transcripts encoding breast disease markerproteins. Preferably, the nucleotide probes hybridize with a targetsequence under high stringency conditions. As used herein, “moderatestringency” and high stringency” refers to finite ranges ofhybridization conditions that are well established in the literature.(See, for example: Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor, N.Y., Cold Spring Harbor Press, 1989; Hamesand Higgins, eds., Nucleic Acid Hybridization: A Practical Approach, IRLPress, Washington D.C., 1985; Berger and Kimmel, eds, Methods inEnzymology, Vol. 52, Guide to Molecular Cloning Techniques, AcademicPress Inc., New York, N.Y., 1987; and Bothwell, Yancopoulos and Alt,eds, Methods for Cloning and Analysis of Eukaryotic Genes, Jones andBartlett Publishers, Boston, Mass. 1990; each of which is incorporatedherein by reference in its entirety. Moderate or high stringencyhybridization conditions are achieved, e.g., by adjusting thetemperature of hybridization, adjusting the percentage ofhelix-destabilizing agents such as formamide in the hybridization mix,and adjusting the temperature and salt concentration of the washsolutions. Alternatively, stringency can be adjusted duringpost-hybridization washes by varying the salt concentration and/or thetemperature. Stringency of hybridization may be reduced by reducing thepercentage of formamide in the hybridization solution or by decreasingthe temperature of the wash solution. Typical high stringency conditionsrequire, for example, high temperature hybridization (e.g., 65-68° C. inaqueous solution containing 4-6× SSC, or 42° C. in 50% formamide)combined with a high temperature (e.g., 5-25° C. below the Tm) wash anda low salt concentration (e.g., 0.1× SSC). In contrast, moderatestringency conditions involve, for example, hybridization at atemperature between 50° C. and 55° C. and washes in 0.1× SSC, 0.1% SDSat between 50° C. and 55° C., which should be sufficient to identifypolynucleotide molecules encoding I-mf from other species or to isolateisoforms of I-mf. In further contrast, low stringency conditionsinvolve, for example, low hybridization temperatures (e.g., 35-42° C. in20-50% formamide) and intermediate temperature (e.g., 406° C.) washes ina higher salt concentration (e.g., 2-6× SSC).

[0110] In certain preferred embodiments of the invention, cDNA andoligonucleotide probes are employed in well known Northern, Southern anddot-blot assays for identifying and quantifying the level of expressionof a selected breast disease marker in cell samples collected fromexpressed mammary fluid.

[0111] Other suitable probes for use within the invention includelabeled ligands, binding partners and co-factors of breast diseasemarkers (e.g. growth factor receptor ligands, or substrates of breastcancer associated proteases such as cathepsin D).

[0112] Measuring the level of expression of breast disease markersaccording to the foregoing methods will provide important prognostic andtreatment-related information for assessing a broad range of breastdisease, including the genesis, growth and invasiveness of cancer, inmammals, particularly humans. For example, assays utilizingoligonucleotide probes will assist early screening to evaluate heritablegenetic lesions associated with breast cancer, and to distinguishbetween precancerous, early cancerous and likely metastatic lesions inpatients.

[0113] In addition to the above mentioned sample collection and assaymethods, the invention also provides kits and multicontainer unitscomprising devices, components, accessories, reagents and other relatedmaterials for practicing the sample collection and assay methods of theinvention. These kits are clinically useful for collecting, handlingand/or processing mammary fluid samples, e.g., for determining thepresence and/or amount of a breast disease marker, preferably a breastcancer marker, in the biological samples. The kits include a mammaryfluid collection device having a breast engaging member, vacuum pump,and sample collection means incorporated in a general purpose orhand-held breast pump as described herein. Additional kits include oneor more breast pump attachments (e.g., a detachable breast engagingmember, or multiple such attachments for use with different patients),accessories (e.g., replaceable fluid-retaining reservoirs), solid phasemedia, and/or disposable or reusable support members, cartridges orcassettes for holding collection media, as described herein. These andother kit components may be provided, alone or in any combination, withor without inclusion of the basic breast pump apparatus in the kit. Yetadditional kits include a pharmaceutical preparation of oxytocin or anoxytocin analog in a biologically suitable carrier for use in alternatemammary fluid collection methods of the invention. Preferably, theoxytocin preparation is provided in an intranasal spray applicator andcontains approximately 40 USP units of oxytocin per ml of liquidcarrier, which carrier is a simple, inexpensive buffered salinesolution. Preferred applicators can be in any of a variety ofpressurized aerosol or hand-pump reservoir forms, with a nozzle fordirecting a liquid spray of the oxytocin into a patient's nostril. Stillother kits include on or more preparative and/or diagnostic reagentsselected from those disclosed herein, including one or more fixatives,probes, labels and the like in separate or common containers. In certainembodiments of the invention, kits include compositions and/or devicesfor detecting the presence or amount of one or more breast diseasemarker(s) in the biological sample, often including one or moreimmunological or molecular probe(s) that binds or reacts with one ormore breast cancer marker(s). The foregoing kit components are generallyassembled in a collective packaging unit, which may include written orotherwise user-accessible instructions detailing the sample collection,handling and/or processing methods of the invention.

[0114] Kits for practicing the assay methods of the invention include asuitable container or other device for collecting, storing, handlingand/or processing a biological sample from expressed mammary fluid. Arange of suitable collection devices is contemplated corresponding to awide range of suitable biological samples that may be collected from theexpressed mammary fluid. For example, simple sterile containers orreservoirs are provided to collect whole mammary fluid. Alternatively, avariety of solid phase devices, including microscopic glass slides,membranes, filters, beads and like media, are provided to receive orpartition selected liquid or solid fractions of the mammary fluid, toreceive or partition cells or cellular constituents from the mammaryfluid, or to receive or partition purified or bulk proteins,glycoproteins, peptides, nucleotides (including DNA and RNApolynucleotides) or other like biochemical and molecular constituentsfrom the mammary fluid. A wide variety of such sample collection devicesare disclosed herein, or are otherwise widely known or described in theliterature, which can be readily adapted for use within specificembodiments of the invention. These collection devices may be providedas a component of the breast pump (such as a removable nitrocellulosefilter placed within the pump, optionally coupled there with by apermanent or removable support member, to directly receive or contactthe expressed mammary fluid as it is pumped), or may be providedseparately (for example as a non-integral membrane, filter, affinitycolumn or blotting material to which mammary fluid or mammary fluidcomponents are exposed to collect a biological sample for assaypurposes). In more detailed aspects, the collection device includes aremovable, fluid-retaining reservoir as described herein below.

[0115] In certain embodiments of the invention, kits include reagentsand/or devices for detecting the presence and/or amount of a breastdisease marker in the biological sample, for example an immunological ormolecular probe that binds or reacts with a breast cancer marker. Amongthese possible reagents immunological and non-immunological probes fordetecting the presence or amount of a breast cancer marker in thebiological sample. The kits may also contain suitable buffers,preservatives such as protease inhibitors, direct or sandwich-typelabels for labeling the probes, and/or developing reagents for detectinga signal from the label. In one aspect, kits of the present inventioncontain monoclonal antibodies useful for detecting and/or measuring abreast cancer marker in a sample. Such antibodies may be pre-labeled, ormay be detected by binding to a secondary antibody optionally includedin the kit. The antibody reagents may be provided in a separatecontainer, or may be provided in combination in a series of containers.Within yet another aspect of the invention, kits containsequence-specific oligonucleotide primers for detecting polynucleotidemolecules encoding breast cancer marker proteins. Such primers may beprovided in separate containers, or may be provided in combinations ofone or more primer pairs in a series of containers. A broad selection ofother kits are provided within the invention based on general knowledgein the art and on the description herein, including kits that containspecific instructions for carrying out the assays of the invention.

[0116] Also provided within the invention are methods for obtaining abiological sample from a patient and/or determining the amount of abreast disease marker in a biological sample from breast fluid, whichmethods employ a novel breast pump 10 or breast pump adapter 12, asdescribed herein below. These methods include a step of applying thebreast pump to induce breast fluid expression, wherein a solid phasesample collection medium is fluidly connected with the breast pump. Thesolid phase sample collection medium may be integrated within the breastpump or otherwise fluidly connected with the pump, so that an expressedbreast fluid sample contacts the collection medium while the pumpremains applied to the breast.

[0117] To practice these aspects of the invention, the breast pump 10(FIG. 1) and breast pump adapter 12 (FIG. 12) each have fluidlyconnected therewith a solid phase sample collection medium selected fromany of the solid phase media described herein above. The breast pump maybe generally constructed according to various conventional breast pumpdesigns, for example according to the general design described in U.S.Pat. No. 4,929,229 and U.S. Pat. No. 5,007,899 to Larsson; U.S. Pat. No.5,601,531 to Silver; U.S. Pat. No. 3,786,801 to Sartorius; or U.S. Pat.No. 5,295,957 to Aidaet al.

[0118] As with other conventional breast pumps, the breast pump 10 ofthe invention includes a breast engaging portion 14 constructed of anon-porous material. The engaging portion is sized and dimensioned toreceive at least a nipple 16 portion of a breast 17 and form a suctionseal therewith. Preferably, the breast engaging portion is sized anddimensioned to receive at least an areolar portion of the breast, andmore preferably a distal quarter to one-half or larger portion of thebreast (e.g., as shown in FIG. 1), and form a suction seal therewith.Different sizes and dimensions of the breast engaging member may beselected, e.g., to receive human breasts of differing sizes.Alternatively, devices for veterinary use are provided wherein thebreast engaging member is sized and dimensioned to receive a breast of anon-human mammal.

[0119] To form a suction seal with the breast 17 as described above, thebreast engaging portion 14 of the pump 10 may be constructed in avariety of shapes and dimensions. In one embodiment the engaging portionis formed as a simple cylinder, tube or funnel shaped and dimensioned toengage the nipple 16 or areolar portion of the breast in a suction seal.Preferably, a terminal edge 18 of the engaging portion is rounded orflared so that the edge does not impinge uncomfortably against the skinof breast 17 when negative pressure is applied to the breast to form thesuction seal. In preferred embodiments the engaging portion is roughlyfunnel shaped to comfortably engage a distal quarter to one-half orlarger portion of the breast, as shown in FIG. 1 and form a suction sealtherewith.

[0120] The breast engaging portion 14 of the breast pump 10 can beconstructed of any suitable non-porous material which is inert to bodyfluids and which has sufficient rigidity to prevent collapse of theengaging portion when negative pressure is applied against its innerwalls 20. Preferably, the engaging portion and other parts of the breastpump are autoclavable for sterilization purposes. Thus, the engagingportion may be constructed of a rigid material such as a polypropylene,polyurethane, polyvinyl plastic, polymethyl methacrylate, and the like.Alternatively, the engaging portion may be constructed of a semi rigidmaterial which prevents collapse but allows for manual compression of atleast a base 22 of the engaging portion to massage the nipple 16 and/orareolar region of the breast 17 to facilitate breast fluid expression.Suitable materials in this context include rubber or syntheticelastomers, e.g., silicon plastic (silastic) and like materials.Preferably, the material that forms the engaging portion is transparentto allow a physician or technician using the breast pump to visualizethe breast 17 to determine its positioning and condition duringapplication of the pump and to observe fluid expression from the nipple.

[0121] The breast engaging portion 14 of the breast pump 10 is fluidlyconnected to a sample collection housing 30 made of a rigid material(preferably transparent plastic). The solid phase sample collectionmedium, as described above, is supported in fluid connection with thebreast engaging portion, for example by anchoring the solid medium to,or within, the breast engaging portion or sample collection housing.Typically, the solid phase sample collection medium is affixed within aninterior compartment or lumen 58 of the sample collection housing orcorresponding, fluidly connected, interior space of the breast engagingportion-using any of a wide range of optional anchoring or positioningmeans. Preferably, the solid phase medium is removably supported influid connection with the breast engaging portion, e.g., by means of acloseable retainer or replaceable cassette (see below).

[0122] In one aspect of the invention the sample collection housing 30or breast engaging portion 14 supports a sample collection pad, orsheet, 38 of absorbent or adsorbent material, for example a membrane 39or filter 40 pad or sheet (FIGS. 2-5). Multiple pads or sheets (of thesame or different material) may be used in combination. For example, amembrane 39 (e.g., nitrocellulose) may be supported on a filter 40(e.g., a paper filter) as shown in FIG. 5. In this manner, a first sheetmay serve as a support member, a wetting member, a wicking member, or apartitioning member for a second sheet, or may introduce or remove achemical reagent, probe, blocking agent, buffering agent, denaturingagent, etc. therefrom. In one aspect, the multiple sheet materialspartition components of the breast fluid (e.g., by using differentmaterials to retain different components of the breast fluid), therebyallowing for collection of different samples simultaneously.

[0123] In another aspect of the invention the housing 30 or breastengaging portion 14 supports a particulate solid phase sample collectionmedium 41, for example beads, resins, microspheres, particulatechromatographic media (e.g., agarose or silicate media), and the like(see, e.g., FIG. 9). In yet another aspect of the invention, the housingor breast engaging portion supports a non-particulate solid template forsample collection, for example one or more capillary tubes 42 (FIG. 6),coated tubes 43 (FIG. 10), plates, wells, slides and the like formed ofglass, plastic or other suitable materials.

[0124] As shown in FIGS. 1 and 2, a preferred design of the breast pump10 includes a removable coupling mechanism between the engaging portion14 and the sample collection housing 30. A preferred coupling mechanismincludes complementary threads 44, 46, disposed at mated connecting ends48, 50 of the engaging portion, and housing, respectively.Alternatively, a simple pressure fit coupling may be provided toremovably couple mated connecting ends 48, 50 of the engaging portionand housing, as shown in FIG. 9. In yet another alternative embodiment,the connecting ends 48, 50 are removably coupled by a hinge 52 and latch54 that pivotally connects the two connecting ends (FIG. 10).

[0125] The sample collection housing 30 or breast engaging portion 14can support the solid phase sample collection medium in several ways, asexemplified in the drawings and by a variety of equivalent designs andconfigurations that will be apparent to the artisan. In preferredembodiments of the invention, the solid phase medium is held on orwithin a support member 56 adapted to support the solid phase medium influid connection with the breast engaging portion, for example a supportthat is fixedly interposed between the engaging portion 14 and thesample collection housing.

[0126] Thus, in one exemplary design shown in FIGS. 1 and 2, the supportmember 56 is a removable disc spanning a lumen 58 of the housing andinterposed between connecting ends 48, 50 of the engaging portion andhousing. For use in conjunction with a variety of breast pump designs, adiameter 59 (FIG. 3) of the support member is between about ¼-3.0inches, preferably about ½-2.0 inches, and more preferably about ¾-1inches. In preferred aspects, the disc-shaped support member seatswithin a circumferential groove 60 in the connecting end 48 of thehousing. A complementary circumferential groove 62 in the connecting end46 of the engaging portion opposes the circumferential groove in theconnecting end of the housing to sandwich the disc-shaped support membertherebetween.

[0127] In this embodiment, prior to connecting the engaging portion 14of the breast pump 30 with the housing 30, the support member is seatedtherebetween (e.g. by fitting the support member within the opposingcircumferential grooves 60, 62 of the housing and engaging portion). Theforce of connection (i.e. threading, pivoting or pushing the engagingportion and housing relative to one another) firmly sandwiches thesupport member in position between the engaging portion and housing.

[0128] To facilitate this purpose, the thickness (i.e., sectionalheight) 63 of the support member 56 in the present embodiment is equalto or slightly greater than the height of a sidewall 64 of thecircumferential groove 60 of the housing 30, whereby the support memberis held in a friction fit and may be partially compressed when theengaging portion and housing are connected. Thus, the thickness of thesupport member is between about 2 mm to 5 cm, preferably about 3 mm to 2cm, and more preferably about 4 mm to 1 cm. Consistent with this design,the support member can be made of a hard plastic material (e.g., a hardpolyvinyl or polyurethane), but is preferably made of a resilient,moderately compressible material, e.g., soft plastic, rubber, or awaterproof fiber or composite material as used in conventional plumbingand automotive gaskets.

[0129] A disc-shaped support member 56 is well suited to support a sheet38 of absorbent or adsorbent material, such as a membrane or filter. Asshown in FIGS. 2 and 7, the sheet is preferably sandwiched between anupper retainer ring 66 and a lower retainer ring 68 of the supportmember to hold the sheet in place against negative pressure that maypass through the filter when a vacuum is applied through the engagingportion 14 and housing 30 (see below), as well as when the nipple 16impinges against the sheet. The upper and lower retainer rings may beintegrally joined in a disposable refill as shown in FIG. 2, or the tworetainer rings may be separable to provide a reusable cassette forremoving and inserting replacement sheets. An example of the latterdesign is depicted in FIG. 7, where the upper and lower retainer ringsare releasably interconnected, e.g., by a hinge 71 or other connectingmeans such as an interlocking threading or detent fit mechanism. In thisembodiment the upper and lower rings can be opened or disconnected toallow insertion and removal of the sheet, and juxtaposingly closed,e.g., by a snap 72 on one ring adapted to form a detent fit within areceptacle 74 on the opposing ring, thereby holding the sheet in a fixedposition between the two rings. To facilitate this purpose, opposingfaces 75 of the upper and lower rings may have a rugose or otherwisedecorated surface to facilitate retention of the sheet, for example aridge 76 or ridges to engage the sheet and securely clamp the sheetbetween the tworings.

[0130] In an alternative design depicted in FIGS. 4 and 5, there is noupper retaining ring 72 and the sheet 38 simply rests upon the supportmember 56 or is removably retained against an upper surface 76 of thesupport member by alternative retaining means. For example, the sheetmay be fitted within a recess 78 surrounding the upper surface of thesupport member that is shaped and dimensioned to receive the sheet. Thesheet may be securely fitted within the recess, e.g., by appropriatelysizing the sheet so an edge of the sheet frictionally engages a sidewall79 of the recess. Alternatively, a retaining groove may be providedbetween the sidewall of the recess and the upper surface of the supportmember to receive the edge of the sheet and thereby retain the sheet bya detention fit within the recess during use. In yet another alternativedesign, the sheet simply rests atop the upper surface of the supportmember and is removably secured thereto, e.g., by wetting or gluing(preferably with an inert bonding agent) to create a temporary bondbetween the sheet and upper support member surface. In each of theforegoing designs, the sheet can be easily seated within or atop thehousing for sample collection and removed thereafter for processing,e.g., by hand or using forceps or other conventional handling tools.

[0131] In preferred embodiments of the invention, the support member 56includes a recess 78 which forms a fluid-retaining well, as shown inFIG. 5. The recess can thus be filled with a desired solution, such as abuffer, a solution containing a probe, cross-linking agent, blockingagent, denaturing agent, etc., to facilitate sample collection,handling, and/or processing.

[0132] Where the design of the support member 56 is such that it spansthe lumen 58 of the sample collection housing 30 or correspondinginterior compartment of the breast engaging member 14, or when thesupport member contains a recess 78 forming a well, it is generallydesirable to provide air channels 80 in the support member 56 to allownegative vacuum pressure to pass from the housing through the airchannels to the engaging portion 14 of the pump during operation, and toallow venting of the engaging portion and housing to permitdisengagement of the engaging portion from the breast 17 after use.Preferably, one or more such air channels are located near the peripheryof the support member, as shown in FIGS. 2, 3, 5, 6 and 9.Alternatively, one or more air channels may be centrally located, asshown in FIG. 4. The air channels may be positioned so that they do notcommunicate with the solid phase sample collection medium, as shown inFIGS. 2, 3, 5, 6 and 9, or they may communicate and form a gaseousconnection therewith (provided that the solid phase medium is porous andhas sufficient strength to withstand vacuum pressures transmittedthrough the air channel), as shown in FIG. 4.

[0133] Alternative designs and configurations of the housing 30, breastengaging member 14, and/or support member 56 are also provided whichvary with the type of solid phase sample collection medium used. Forexample, when a particulate solid phase sample collection medium 41(e.g. beads, resins, or microspheres) is used, the medium may beenclosed in a cartridge 82 removably mounted to, or integrated within,the support member or otherwise removably connected to the samplecollection housing 30 or breast engaging portion. As shown in FIG. 9,preferred embodiments of the invention provide a removable engagementmechanism which allows the cartridge or other receptacle containing thesolid phase medium to be removably engaged relative to the housing orbreast engaging member, e.g., by engaging the cartridge with a supportmember so that a first end of the cartridge makes a fluid connectionwith the engaging portion 14 of the pump 10. In one embodiment, thefirst end of the cartridge is removably inserted through a mountingchannel 86 that passes through the support member to provide a fluidconnection between the engaging portion of the pump and the cartridgefirst end. Preferably, the channel is dimensioned to receive the firstend of the cartridge in a friction fit (e.g., wherein a diameter of thechannel is about 0.5 mm to 2 cm, preferably about 1 mm to 1 cm, and morepreferably about 3-5 mm), whereby the cartridge can simply be pushedinto the channel until the cartridge first end is flush with, or extendsslightly above, the upper surface 76 of the support member and willremain in place during use. For this purpose it is also preferable toform at least the channel portion of the support member from aresilient, moderately compressible material so that the channelyieldingly receives and releases the cartridge in a moderate (i.e.,readily hand removed) friction fit. Alternatively, the cartridge can beengaged relative to the housing by complementary threading orinterlocking detent fitting (e.g. a conventional key and groove design)between the cartridge first end and the support member channel). In yetother alternative designs the cartridge can be permanently engaged withthe support member or engaged directly to the housing.

[0134] Design and construction of the cartridge 82 will vary dependingon the characteristics of the particulate solid phase medium used,including the size of the particles, the function of the particles(e.g., chromatography adsorption, affinity binding, etc.), and whetherthe particles are used dry or are contained in a solution, among otherfactors. Design and construction of the cartridge will further depend onthe type of breast diseasemarker(s) which may be sought for detection inthe sample (e.g., cells, proteins, lipids or nucleic acids).

[0135] In a preferred embodiment shown in FIG. 9, the cartridge iscylindrical and contains beads or microspheres. To enclose the beads ormicrospheres in the cylinder while maintaining a fluid connection withthe engaging portion 14 of the pump 10, the first end 84 of the cylinderis covered by a semi-permeable cover 90 of a porous barrier material(e.g, a filter or membrane) which allows breast fluid (including orexcluding selected components of the fluid, such as cells) to passthrough the cover to contact the beads or microspheres, while preventingescape of the beads or microspheres from the cartridge. In this manner,the cover can partition components of the breast fluid into thecartridge, and can also separately retain different components on thecover, thereby allowing for collection of different samplessimultaneously. The semi-permeable cover can be affixed to the cartridgeby a variety of means, e.g., by bonding with a removable or permanentbonding agent, or by providing a removable or integral cover retainingring 92 to secure the cover to the cartridge first end 84. A second endof the cartridge features a second end cover 96 which may be integral toor removable from the cartridge, and which may be impermeable to gas andfluids or semi permeable as described above for the first end cover.

[0136] In another aspect of the invention, the housing 30 supports anon-particulate solid template for sample collection. This type of solidphase collection medium includes, e.g., one or more capillary tubes 42(FIG. 6), coated tubes 43 (FIG. 10), plates, wells, slides and the like.These templates for receiving, adsorbing or binding a sample of breastfluid (or desired components thereof) are preferably formed of glass,plastic or like materials known in the art to be suitable for samplecollection (e.g. inert plastics).

[0137] To accommodate these various templates, yet additionalalternative designs and configurations of the housing 30, breastengaging portion 14, and/or support member 56 are provided. For example,when capillary tubes 42 are used, these may be mounted to or integratedwithin the support member, or anchored by a variety of other comparablemeans with respect to the housing 30. As shown in FIG. 6, preferredembodiments of the invention utilize a support member with one or moremounting channels 86 to removably receive a first end of one or morecapillary tubes 42, so that the end of the tube makes a fluid connectionwith the engaging portion 14 of the pump 10. Thus, the channels have apreferred diameter equal to or slightly less than a diameter of astandard capillary tube, i.e., about 0.5 mm to 3 mm, preferably about1-2 mm and more preferably about 1.5 mm. Construction of the supportmember and mounting of the tube(s) is similar to support memberconstruction and mounting of the cartridge as described above. When asingle tube is used, it is preferably placed centrally relative to thehousing. When multiple tubes are used they may be arrayed to collectmultiple samples simultaneously, e.g., as shown in FIG. 6.

[0138] Another alternative solid template for sample collection providedwithin the invention is a coated tube 43 which is preferably mountedrelative to the housing 30 in the same manner as described above forcapillary tubes 42 (FIG. 10). The tube may be open at both ends, or mayhave a semi-permeable cover at one or both ends, as well as animpermeable second end cover, as described above for the cartridge 82.

[0139] The coated tube has a lumenal coating 100 adapted for adsorbing,binding, partitioning or otherwise processing the breast fluid sample.For example, the coating may be an affinity coating having an antibody,ligand, or other binding partner that specifically binds a selectedbreast disease marker, wherein the coating is covalently or otherwisebound to a lumenal wall of the tube. A wide variety of useful coatingsare disclosed herein or are otherwise well known in the art. Thesecoatings may also be used to coat other solid phase media for use withinthe invention, including templates such as wells, plates, slides, etc,including a well formed by a recess 78 in a support member 56.

[0140] Because only small droplets of breast fluid will typically beexpressed at the surface of the nipple 16, it is generally preferred todirectly contact the expressed fluid on the nipple with the solid phasesample collection medium. This requires positioning of the samplecollection medium close to the base 22 of the breast engaging portion 14of the pump as shown in the figures. Thus, when a support member 56 isprovided it is positioned so that its upper surface 76 will directlycontact the nipple during application of negative pressure through theengaging portion to the breast. Only approximate positioning isgenerally required in this regard, because the nipple will tend to bedrawn toward the support member by the vacuum and thereby will abut theupper support member surface.

[0141] However, because breast pump designs and breast anatomy varysignificantly, it is preferable to adjustably mount the solid phasemedium relative to the housing 30 so that it can be moved closer to, orfarther away from, the base 22 of the engaging portion 14 of the pump10. Thus, in preferred embodiments of the invention a reciprocatingmechanism is provided which adjustably moves the solid phase collectionmedium in closer, or more distant, proximity to the nipple when thebreast pump is engaged therewith. At the beginning of the fluidexpression procedure, the collection medium is retracted away from thenipple while negative pressure is applied to the breast to facilitatefluid expression. Fluid expression is visualized through a transparentengaging portion or housing, and the collection medium is then advancedproximal to the nipple to contact the expressed fluid.

[0142] As shown in FIG. 11, a preferred design for the reciprocatingmechanism incorporates a support member 56 to support the solid phasecollection medium, as described above. The support member isreciprocatingly mounted relative to a rotating member 109 of the housing30, preferably on a reciprocating carrier 110. The support member may beremovably mounted to the carrier, e.g., by friction fitting, detentionfitting or threadedly engaging the support member to a first end 112 ofthe carrier, as described above for mounting the support member to thehousing and/or engaging portion 14 of the pump 10. For example, thesupport member may be mounted by friction fitting within acircumferential groove 114 at the first end of the carrier. Inconjunction with this design, the carrier is preferably in the form ofan open cylinder so that negative pressure can be effectivelytransmitted through the carrier and support member to the engagingportion.

[0143] To reciprocatingly adjust the position of the carrier 110 and/orsupport member 56 relative to the engaging portion 14 of the pump 10,the rotating member 109 of the housing 30 is sealably, rotatably, andremovably interconnected to the base 22 of the engaging portion. Thisinterconnection may be accomplished by a variety of designs, one ofwhich is to seat a first O-ring 116 in opposing circumferential grooves118, 120 in the connecting ends 48, 50 of the engaging portion, and therotating member of the housing, respectively. These grooves are sizedand dimensioned to receive the O-ring in an airtight seal when vacuumpressure is applied through the housing and engaging portion of thepump, without substantially compressing the O-ring. The O-ring is alsolubricated, e.g, with silicon grease. These features allow free rotationof the rotating member of the housing relative to the engaging portionof the pump, which rotation drives the reciprocating mechanism toadvance the sample collection medium (e.g., by advancing the carrierand/or support member) to contact the expressed breast fluid on thenipple 16.

[0144] To complete the reciprocating mechanism for the above describedembodiment of the invention, the rotating member 109 of the housing 30is also sealably and rotatably interconnected to a stationary member 124of the housing. This interconnection is preferably achieved by seating asecond O-ring 126 in opposing circumferential grooves 128, 130 in a rearconnecting end 132 of the rotating member of the housing and a frontconnecting end 134 of the stationary member 124 of the housing,respectively. These grooves are also sized and dimensioned to receivethe O-ring in an airtight seal without substantially compressing theO-ring, and the O-ring is lubricated to facilitate free rotation of therotating member relative to the stationary member.

[0145] To reciprocate the carrier 110 and/or support member 56 forwardand backward relative to the engaging portion 14, the rotating member109 of the housing 30 is provided with a lumenal, helically orientedgroove 140 dimensioned to receive a riding peg 142 extendingtransversely from the carrier or support member. In addition, therotating member of the housing is provided with a longitudinallyoriented, lumenal groove 144 dimensioned to receive an angularlyfixating keel 146 extending transversely from the carrier or supportmember. In accordance with this design, rotation of the rotating member109 of the housing 30 drives rotation of the carrier or support memberwhich is angularly fixed relative to the rotating member by the fixatingkeel engaged with the longitudinal groove of the rotating member. As therotating member of the housing and carrier thus rotate (with theposition of the engaging portion and stationary member of the housingangularly fixed by friction or manual or structural resistance), theriding peg rides along the helical groove, translating the peg in thedirection of the groove and thereby causing the support member orcarrier to reciprocate forward or backward relative to the engagingportion.

[0146] To insert and remove the solid phase medium and/or support member56 from the rotating member 109 of the housing 30, a removableinterconnection is provided between the rotating member and the base 22of the engaging portion, as described above. To uncouple the rotatingmember and engaging portion, all that is required is that these parts bepulled in opposing directions, whereby the O-ring 116 will unseat fromone of the opposing circumferential grooves 118, 120 in the connectingends 48, 50 of the engaging portion and rotating member, respectively.To recouple the rotating member and engaging portion after loading orretrieval of the sample collection medium and/or support member, theyare simply pushed back together. To facilitate reseating of the O-ring,it may be desired to make one of the opposing circumferential groovesdeeper than the other, so that the deeper groove retains the O-ring whenthe rotating member and engaging portion are separated, and theshallower groove more readily accepts the O-ring when they arere-coupled.

[0147] An alternative reciprocating mechanism is provided within theinvention which uses a simple slide mechanism to reciprocate the samplecollection medium relative to the engaging portion 14 of the pump 10, asshown in FIGS. 14 and 15. One embodiment of the slide mechanism featuresa manifold 150 defining an inner lumen 152 that is not in gaseousconnection with an outer lumen 154 of the housing. This design providesfor a manual slide lever 156 to extend to the outside of the housing sothat a head portion 158 of the lever can be manually engaged by a pumpoperator. The slide lever is in turn connected to the support member 56or carrier 110 which are sized and dimensioned to allow the carrier toreciprocate freely within the inner lumen.

[0148] In operation, the slide lever 156 is moved to a rearward positionso that the solid phase sample collection medium (e.g., a pad or sheet38 of absorbent material is out of contact with the nipple 16, as shownin FIG. 14. Negative pressure is applied through the outer lumen 154 tothe area of the breast surrounding the nipple, the tip of which isaligned with the inner lumen. Breast fluid expression is visualizedthrough the transparent engaging portion and housing, at which time thelever is manually engaged by the head portion 158 and moved forward.Movement of the lever causes the support member and/or carrier to moveforward until the sample collection medium contacts the expressed fluidat the tip of the nipple. The engaging portion and housing are removablyconnected, e.g., by a hinge 52 and latch 54 or other suitable connectionmeans, thereby allowing for easy insertion and removal of the solidphase medium and/or support member.

[0149] In each of the foregoing breast pump designs, the engagingportion 14 of the breast pump 10 is in gaseous connection with a vacuumpump 160 capable of generating sustained negative pressure in an area ofthe breast 17 surrounding the nipple 16 (see FIG. 1). Any of a largevariety of vacuum pumps, which are well known for use in conjunctionwith breast pumps, can be used, including manual pumps (FIG. 1),mechanically driven pumps and electrically driven pumps. When activated,the pump generates negative pressures of between about 50-200 mm Hg.Typically the pump will be connected via a heavy vacuum hose 162 inconnection with the engaging portion. Generally, the hose is connectedto the housing 30 which will is in gaseous connection with the engagingportion (see, e.g, FIGS. 1, 8 and 11).

[0150] Pressure exerted upon the breast 17 by the pump can be varied inaccordance with well known pressure modulating mechanisms (e.g., byproviding a diaphragm or other mechanism to modulate a diameter of an inline, pressure modulating valve). In addition, the breast pump 10includes a venting mechanism, e.g., a pressure release valve 164, whichthe user can selectively operate to close and vent the system before andafter use, thereby selectively applying and releasing the vacuumpressure acting on the breast. In this regard, the system is generallyvented as soon as sufficient breast fluid expression is observed by theoperator. This also relieves pressure on seals (e.g., O-rings 116, 126),when the reciprocating mechanism relies on a sealable and rotatableconnection between different parts of the pump (as in FIGS. 11 and 13),thereby facilitating respective rotation of the different parts toreciprocate the support member 56 and/or carrier 110.

[0151] In yet another aspect of the invention, a breast pump adapter 12is provided which couples a solid phase sample collection medium with aconventional breast pump (See FIGS. 12 and 13). As shown in FIG. 12, theadapter features a replacement breast engaging portion 170 sized anddimensioned for removable insertion within a breast engaging portion 14of a conventional breast pump. In preferred embodiments, the replacementbreast engaging portion is funnel shaped and nests within a funnelshaped breast engaging portion of an existing breast pump. When fullynested, a terminal edge 172 of the replacement breast engaging portionextends at least as far as the terminal edge 18 of the breast engagingportion of the existing breast pump.

[0152] The replacement breast engaging portion 170 can be removablyconnected to the breast engaging portion 14 of the existing breast pump10 by a variety of means, e.g, by friction fitting, detention fitting orthreadedly engaging the replacement engaging portion with the breastengaging portion of the existing pump. Preferably, the adapter 12 has astem portion 174 that extends into a cylindrical, connecting portion 176of the existing pump, and the stem portion cooperates with this part ofthe existing pump to provide a removable connection mechanism. Thus, inone preferred embodiment the stem portion features a circumferentialgroove 178 dimensioned to receive an O-ring 180, which O-ring impingesagainst an inner wall 182 of the connecting portion to create a frictionfit to interconnect the replacement engaging portion with the breastengaging portion of the existing pump.

[0153] The adapter 12 supports a solid phase sample collection medium influid connection with the replacement engaging portion 170. Preferably,the solid phase medium is connected with the replacement engagingportion by a support member 56, as described above. The support membermay be integrally or removably mounted to the adapter, e.g., by frictionfitting, detention fitting or threadedly engaging the support member tothe stem 174 of the replacement engaging portion, in a position thatwill allow contact between the nipple and solid phase medium during orafter breast fluid expression. For example, the support member may bemounted by friction fitting within a circumferential groove 184 at abase of the stem (FIG. 12).

[0154] In preferred embodiments of the breast pump adapter 12, areciprocating mechanism is provided to move the solid phase samplecollection medium relative to the replacement engaging portion 172, inaccordance with the concepts described above. As shown in FIG. 13, apreferred design for the adapter having a reciprocating mechanismfeatures a replacement breast engaging portion 170 sealably androtatably nested within a rotating dial member 190, which is in turnsized and dimensioned for removable insertion within a breast engagingportion 14 of an existing breast pump. The replacement breast engagingportion and rotating dial member are preferably funnel shaped tocollectively nest within a funnel shaped breast engaging portion of aconventional breast pump. When fully nested, a terminal edge 172 of thereplacement breast engaging portion and free edge 192 of the rotatingdial member extend at least as far as the terminal edge 18 of the breastengaging portion of the existing breast pump.

[0155] The rotating dial member 172 is connected to a rotating member109 of the housing 30, preferably as a unitary insert, whereby manualrotation of the dial member drives rotation of the rotating member ofthe housing. The rotating member of the housing is in turn rotatablycoupled with an anchoring member 194 of the housing which anchors theentire housing within the existing pump, e.g., within a cylindrical,connecting portion 176 of the existing pump. As shown in FIG. 13, theanchoring member of the housing is preferably in the form of a sleevethat partially surrounds the rotating member of the housing and issealably, rotatably connected therewith. The anchoring member is in turnnon-rotatingly anchored within the cylindrical, connecting portion ofthe existing pump.

[0156] In one preferred embodiment the rotating member 109 of thehousing is sealably, rotatably connected with the anchoring member 194of the housing by seating a first O-ring 196 in opposing circumferentialgrooves 198, 200 at front connecting ends 202, 204 of the rotatingmember and the anchoring member 194 of the housing, respectively. Thesegrooves are sized and dimensioned to receive the O-ring in an airtightseal between the rotating member and anchoring member, withoutsubstantially compressing the O-ring. The O-ring is also lubricated tofacilitate free rotation of the rotating member relative to theanchoring member. A second, lubricated and non-compressingly seatedO-ring 206 is seated in opposing circumferential grooves 208, 210 atrear connecting ends 212, 214 of the rotating member and anchoringmember of the housing, respectively, to facilitate rotation of therotating member relative to the anchoring member.

[0157] To align and facilitate rotation of the rotating member 109 ofthe housing, the rotating dial member 190 (which drives the rotatingmember of the housing) is sealably, rotatably connected with thereplacement engaging portion 170 of the adapter 12. Preferably, thereplacement engaging portion has a stem 216 which nests within astem-shaped base 218 of the rotating dial member. Free rotation betweenthese structures is achieved, e.g., by providing a third lubricated andnon-compressingly seated O-ring 220 seated in opposing circumferentialgrooves 222, 224 in the stem and base of the replacement engagingportion and rotating dial member, respectively. This rotation is alsofacilitated by friction contact (by pressure and/or suction) between thereplacement engaging portion and the breast 17 of the patient, whichangularly secures the replacement engaging portion and prevents itsco-rotation with the rotating dial member.

[0158] The anchoring member 194 of the housing is in turn anchoredwithin the existing pump by an anchoring mechanism that angularlysecures the anchoring member within the pump, e.g., against an innerwall 182 of the cylindrical connecting portion 176. For example, frontand rear compressible anchoring sleeves 230, 232 may be mounted in frontand rear circumferential anchoring sleeve retainer grooves 234, 236surrounding the anchoring member. The anchoring sleeves arenon-lubricated and are made of a semi-compressible material such asrubber or soft plastic. This construction creates a friction anchorbetween the anchoring member and the inner wall of the connectingportion, so that the anchoring member does not move angularly duringrotation of the rotating member 109 of the housing. Both the anchoringsleeves and retainer grooves are preferably sharply angled at a positioncorresponding to the bases of the retainer grooves (i.e., they have arectangular or triangular cross-section), to securely retain theanchoring sleeves in the grooves despite strong friction against theinner wall of the connecting portion when the anchoring member of thehousing is being inserted into the connecting portion of the existingbreast pump 10 to assemble the adapter 12 with the pump.

[0159] Because the replacement engaging portion 170 is anchored byfriction against the breast 17, and the anchoring member 194 of thehousing 30 is anchored by friction against the inner wall 182 of theconnecting portion 176 of the existing pump 10, the rotating member 109of the housing rotates freely with respect to both the replacementengaging portion and the anchoring member when an operator manuallyengages the rotating dial member 190 and turns it gently whilemaintaining pressure against the breast.

[0160] Relative rotation between the rotating member and anchoringmember of the housing drives the reciprocating mechanism within theinstant embodiment of the invention to advance the sample collectionmedium (e.g., by advancing a carrier 110 and/or support member 56supporting the medium) toward the replacement engaging portion 170 tocontact the expressed breast fluid on the nipple 16. As with previouslydescribed embodiments, the housing 30 preferably houses a support member56 to support the solid phase collection medium, as described above. Thesupport member is reciprocatingly mounted relative to the anchoringmember 194 of the housing 30, preferably on a reciprocating carrier 110.The support member may be removably mounted to the carrier, e.g., byfriction fitting, detention fitting or threadedly engaging the supportmember to a first end 112 of the carrier, as described above. In theembodiment shown in FIG. 13, the support member is removably engagedwith the carrier by cooperative threading 140 between the support memberand carrier. In addition, the support member may be sized anddimensioned for receipt within the stem 216 of the replacement engagingportion, because the replacement engaging portion and an inner (i.e.,lumenal) diameter of the stem thereof are smaller than respectivedimensions of the original engaging portion 14 and its base 22, so thatthe nipple may not fully extend through the stem to contact thecollection medium within the housing. Also in conjunction with thisdesign, the carrier is preferably in the form of an open cylinder andthe rotating member 109 of the housing has a vacuum port 242 so thatnegative pressure can be effectively transmitted through the rotatingmember and carrier (and/or through air channels 80 of the supportmember) to the replacement engaging portion.

[0161] To reciprocatingly adjust the position of the carrier 110 and/orsupport member 56 relative to the replacement engaging portion 170 ofthe adapter 12, the anchoring member 194 of the housing is provided witha lumenal, helically oriented groove 140 dimensioned to receive a ridingpeg 142 extending transversely from the carrier or support member. Inaddition, the rotating member of the housing is provided with alongitudinally oriented, lumenal groove 144 dimensioned to receive anangularly fixating keel 146 extending transversely from the carrier orsupport member. Lastly, the rotating member is provided with a second,longitudinally oriented, lumenal groove 244 to allow access of theriding peg through the wall of the rotating member of the housing intothe helically oriented groove and to allow reciprocating passage of thepin along the groove.

[0162] In accordance with this design, rotation of the rotating dialmember 190 drives rotation of both the rotating member 109 of thehousing 30 as well as the carrier 110 (or support member) which isangularly fixed relative to the rotating member by the fixating keel 146engaged with the longitudinal groove 144 of the rotating member. As therotating member and carrier thus rotate (with the position of thereplacement engaging portion 170 and anchoring member 194 angularlyfixed by friction or manual or structural resistance), the riding pegrides along the helical groove 140, translating the peg in the directionof the groove and thereby causing the support member or carrier toreciprocate forward or backward relative to the replacement engagingportion.

[0163] To insert and remove the solid phase medium and/or support member56 from the adapter 12, removable connections can be uncoupled betweenthe existing pump 10 and the entire adapter unit, between the rotatingmember 190 and anchoring member 194 of the housing, or between therotating dial member and replacement engaging portion 170, among otheraccess designs which will be readily apparent to those skilled in theart.

[0164] In more detailed aspects of the invention illustrated in FIGS.16-26, selected features of a general purpose breast pump 10 for mammaryfluid sample collection as described above are incorporated within ahand-held breast pump device 10′—adapted for greater fidelity and easeof sample collection. The hand-held sample collection pump is uniquelydesigned and constructed to incorporate the breast engaging member 14and the vacuum pump mechanism 160 in a compact, structurally integratedbreast fluid collection apparatus that can be manipulated and operatedwith one hand. As with the general-purpose breast pump 10, describedabove, the handheld pump 10′ incorporates the vacuum pump mechanism 160in gaseous connection with the breast engaging element 14 to routesuction pressure from the pump mechanism, through the engaging element,to apply negative pressure in the area of the nipple 16 of the patient.The solid phase sample collection medium (e.g., a membrane, filter,particulate medium, and/or a non-particulate solid collection templatesuch as a plastic or glass tube, well, vial or slide) is likewisefluidly connected with the breast engaging member—to provide for director indirect transmission of the expressed breast fluid through theengaging member to contact the solid phase collection medium.

[0165] According to these aspects of the invention, novel breast fluidsample collection methods are provided wherein a doctor, technician orpatient collecting a breast fluid specimen can grasp and operate thehand-held breast pump 10′ to stimulate expression of the breast fluidand collect a specimen thereof while keeping one hand free foradditional tasks, such as monitoring the patient and recording patientinformation. In this regard, the compact pump design allows the deviceto be picked up and manipulated with one hand-to seat the breastengaging element against the breast of the patient and thereafter applyvacuum pressure to the breast by manual operation of the vacuum pump 160to stimulate expression of breast fluid. This action causes a suitablevolume of breast fluid to be expressed at or near the nipple 16 forsample collection. In conjunction with these simple operation steps, thehand-held device also allows for simultaneous collection of theexpressed breast fluid onto, or within, the solid phase samplecollection medium that is fluidly connected with the engaging member,often without additional manual steps or a need to remove the devicefrom the breast or otherwise engage two hands in the operation.

[0166] The hand-held breast pump device 10′ can be employed forcollection of breast fluid following oxytocin stimulation to facilitatebreast fluid expression, as described above. Alternatively, the devicecan be used without oxytocin priming to achieve breast fluid expressionby vacuum pressure alone, optionally coupled with mechanical breaststimulation, in a substantial percentage of subjects.

[0167] In using the hand-held breast pump 10′ of the invention expressedbreast fluid is typically transferred directly upon expression to thesolid phase sample collection medium, without intervening manual stepsor a requirement to remove the breast engaging member 14 from the breast17 before the sample is collected. Sample collection in this manner israpid and simple, and promotes sanitary application of the device tominimize the risk of patient infection and sample contamination. Thus,within certain methods of use for the hand-held breast pump, breastfluid is directly transferred to a solid phase collection medium, forexample a membrane, filter, reservoir or vial, integrated within thehand-held pump. The sample may be collected as whole, undiluted breastfluid containing constituent proteins, particulates and/or cells.Alternatively, selected components of the expressed breast fluid may besimultaneously or subsequently removed from the fluid (e.g., byfiltering, partitioning, or refining the breast fluid) to yield aprocessed fluid sample and/or to obtain a solid phase constituentsample. For example, various collection methods are provided which yieldseparated solid components (e.g., cells or other particulates) from thefluid. Alternate collection methods yield soluble, suspended, or solidphase captured proteins, lipids, carbohydrates, polynucleotides or othermolecular/biochemical components from the expressed breast fluid. Incertain embodiments, the hand-held pump device functions to separate orpartition a desired protein, lipid, carbohydrate, or polynucleotidesample material into a solid phase collection medium, such as amembrane, filter, or chromatographic substrate (e.g., leptin-,antibody-, enzyme-, or ligand-coated vials, beads, etc.) In this regard,various alternative or additional steps from knowncollection/chromatographic methods can be employed during samplecollection and processing according to the invention. In this manner,proteins, lipids, carbohydrates, polynucleotides, cells, and noncellularparticulates may be partitioned from liquid components of the breastfluid, or separated from one another, simultaneous with or subsequent toexpression of the fluid, by various known methods, including membraneadsorption, filtering, affinity chromatography, chemical processing,centrifugation, etc. to yield a range of constituent or processedsamples.

[0168] In certain collection methods of the invention, breast fluidexpressed by use of the hand-held pump 10′ is simultaneously orsubsequently diluted, filtered, washed, admixed with fixative or otherprocessing agents, or otherwise processed or modified to yield acollected fluid sample partially or completely devoid of cells, proteinsand/or other selected components originally present in the expressedfluid, to provide a processed fluid sample for laboratory analysis. Inother embodiments, particulate components of the breast fluid, forexample, cells, cellular components and/or cellular debris, arecollected after processing and/or modification, e.g., for cytologicalexamination. These and other alternative collection methods involvingpreliminary sample processing in conjunction with use of the hand-heldbreast pump 10′ are optionally performed simultaneous with, or closelyfollowing expression of the breast fluid. Often, sample collection iscoincident with the fluid contacting one or more solid phase collectionmedium(a) fluidly connected with the breast engaging member 14.Depending on the type(s) of medium(a) used, preliminary sampleprocessing can also be achieved directly by simple operation of thepump, without the need for additional processing steps or removal of thebreast engaging member 14 from the subject's breast. For example, theexpressed breast fluid may be sequentially transferred through multiplemedia, e.g, through a filter or membrane into a liquid-retainingreservoir or container, thereby separating certain components forfurther processing or analysis. Alternatively, the expressed fluid maybe partially processed coincident with transfer to the solid phasemedium(a) by chemical or physical reaction (e.g., adsorption, covalentor affinity linkage, enzymatic reaction, etc.) with the medium or acoating or secondary processing agent admixed or linked therewith.

[0169] In yet other alternative methods within the invention,preliminary sample processing involves additional steps following breastfluid expression. In certain embodiments, the breast engaging member 14is removed from the breast after the breast fluid is expressed and thefluid is transferred to a first solid phase sample collection medium,typically a membrane or filter. This initial or primary stage of samplecollection may be followed by washing or by manual transfer of selectedbreast fluid components (e.g., proteins, carbohydrates, cells, orcellular debris) from the first solid phase collection medium (e.g., anitrocellulose membrane 39) to a second solid phase medium, e.g., aglass slide or fluid-containing reservoir. Typically, preliminary sampleprocessing in this regard precedes final packaging of the collectedsample for storage or shipment to a lab for further processing andanalysis of the sample.

[0170] In more detailed embodiments of the invention, cells or othercellular materials useful for cytological examination are separated orpartitioned simultaneous with or sequential to breast fluid expressiononto or within a first solid phase collection medium. In one example,whole cells are separated from the expressed fluid onto a nitrocellulosemembrane 39 or a filter 40, which is typically secured in fluidconnection with the breast engaging member 14 by a fixed or removablesupport member 56 mounted to the engaging member or sample collectionhousing 30 or otherwise integrated with the hand-held breast pump 10′.The cells are subsequently transferred or washed in fluid (e.g.,cytology fluid) to a second solid phase sample collection medium (e.g.,a slide, well, tube or vial), which may also be connected to, orintegrated with, the breast engaging member or sample collection housingas described.

[0171] To facilitate sample collection according to the foregoingembodiments of the invention, the hand-held breast pump 10′ is typicallyprovided as a compact, hand-held unit for ease of use and convenience ofstorage. As depicted in FIG. 16, certain embodiments of the hand-heldpump comprise a modular device formed of a plurality of components thatare joined or securable in fixed structural interconnection with oneanother. These components, which include a breast engaging member 14,vacuum pump 160 and solid phase sample collection component(s), may bepartially or completely disassembled to remove or uncouple theindividual components, or parts thereof, as desired for efficientoperation, cleaning, servicing and/or storage.

[0172] As described above for the general-purpose breast pump 10 of theinvention, the breast engaging portion 14 of the hand-held pump 10′ isconstructed of a rigid or semi-rigid, non-porous material and is sizedand dimensioned to receive at least the nipple 16 of the subject'sbreast 17 and form a suction seal therewith (see, e.g., FIG. 16). Thebreast engaging portion may be constructed in a variety of shapes anddimensions to accommodate variations in breast anatomy. As alsodescribed above, the terminal edge 18 of the engaging portion is roundedor flared so that the edge impinges comfortably and forms an effectivesuction seal against the skin when negative pressure is applied to thebreast. Typically, the engaging member is constructed of a rigid plasticmaterial which is transparent to allow the operator to visualize thebreast, determine positioning of the device, and observe expression offluid from the nipple 16. Preferably, the engaging portion and otherreusable components of the pump are autoclavable for sterilizationpurposes.

[0173] In more detailed embodiments of the invention shown in FIGS.16-18, the hand-held breast pump 10′ is a modular device comprised ofmultiple, integrated components that are fixedly joined to one anotherwhen the pump is assembled, but can be readily detached or uncoupledfrom one another. This modular configuration of the device allows forinterchanging of parts to adapt the pump for different patients andcollection modes, and to facilitate storage, cleaning and/or servicingof the device. Thus, in one embodiment, the breast engaging portion 14of the pump is provided as a separate, funnel shaped component that isdetachable from one or more interconnecting components of the device(see, e.g., FIG. 18). In this manner, the engaging member can be removedfrom the rest of the device for cleaning and sterilization, or to allowfor interchanging of different engaging members to accommodate breastanatomy differences among patients. Typically, the breast engagingmember is removably coupled with a surface or member of the samplecollection housing 30.

[0174] As illustrated in FIG. 18, the hand-held breast pump 10′ of theinvention incorporates the solid phase sample collection medium in fluidconnection with the breast engaging member 14, typically by use of asupport member 56 affixed to, or removably connected with, the engagingmember. In selected embodiments, the support member encloses or supportsone or more pads or sheets of absorbent or adsorbent material, forexample a nitrocellulose membrane 39. Multiple pads or sheets of thesame or different material may be used in combination (e.g., including awetting member, a wicking member, or a partitioning member).Alternatively, the support member can incorporate or support aparticulate solid phase sample collection medium, for example beads,resins, microspheres, particulate chromatographic media (e.g., agaroseor silicate media), and the like. In yet additional aspects of theinvention, the support member engages or supports a non-particulatesolid template for sample collection, for example one or more capillarytubes, coated tubes, plates, wells, slides and the like formed of glass,plastic or other suitable materials. In certain embodiments, the supportmember may incorporate a compartment, well or reservoir to receive orintroduce sample processing agents selected from chemical reagents,probes, blocking agents, buffering agents, denaturing agents.

[0175] The support member 56 for use in conjunction with the hand-heldbreast pump 10′ is typically provided as a removable cassette that canbe inserted within the engaging member 14, often to seat against theinner wall 20 thereof (see, e.g., FIGS. 16 and 18. In preferred aspects,the support member seats by a friction or compression fit against theinner wall of the engaging member, which may be facilitated by acomplementary circumferential ridge and groove design between thesupport member and engaging member inner wall, as shown in FIG. 16.

[0176] In certain embodiments of the hand-held breast pump, adisc-shaped support member 56 is provided to support one or more sheetsof absorbent or adsorbent material, such as a nitrocellulose membrane39, in close proximity, or in contact with, the nipple 16 when thedevice is in use and suction is applied to the breast. As shown in FIGS.16 and 18, the sheet is preferably secured by an upper retainer ring 66fixedly or removably seated against an opposing surface (exemplified bya circumferential retainer groove 270) with the margin of the sheetsandwiched therebetween. This holds the sheet in place against negativepressure that may pass through the sheet when vacuum pressure is appliedthrough the engaging member and to secure the sheet in position when thenipple impinges against it. The retainer ring may be integrally joinedwith the support member which may comprise a disposable refill, or thering may be separable and the support member may comprise a reusablecassette for receiving replacement sheets. In an alternative design,there is no upper retaining ring and the membrane 39 or filter 40 simplyrests upon the support member 56 or is removably coupled directlytherewith (e.g., by wetting or gluing to create a temporary bond betweenthe sheet and upper support member surface, or by appropriately sizingthe sheet so that a peripheral edge of the sheet engages a surface ofthe support member in a friction or detent fit. Typically, the sheet isreadily removable from the support member for processing, e.g., by handor by using forceps or other conventional handling tools to disengagethe filter or membrane.

[0177] Typically, the support member 56 for use with the hand-heldbreast pump device 10′ includes air channels 80 that pass through thebody of the support member to allow vacuum pressure applied from thevacuum pump 160 to reach the engaging member 14 of the pump duringoperation (i.e., by passing from the pump through the sample collectionhousing 30 and air channels to the engaging member). Likewise, the airchannels allow venting of the engaging member for disengagement from thebreast 17 after use. In certain embodiments, a plurality of two-three ormore air channels are provided, which may be centrally located relativeto a disc-shaped body of the support member as depicted in FIG. 18. Theair channels can serve a dual purpose as channels for passage ortransfer of fluids and/or fluid-suspended particles, including cells andcellular components, between the breast engaging member and the samplecollection housing 30.

[0178] In one related aspect of the invention, cellular materials fromexpressed breast fluid are first collected on a primary solid phasesample collection medium (e.g., a membrane 39 or filter 40) mounted infixed relation to the engaging member 14 (e.g., by a support member 56).In a secondary sample collection or processing step, the cellularmaterials are removed or flushed from the primary sample collectionmedium into a secondary solid phase sample collection medium (e.g., asolid phase sample collection template such as a plastic or glass slide,slip, tube, well or vial), optionally coupled with the support memberand/or sample collection housing. The cellular materials can be directlytransferred from the primary medium onto or into the secondary medium,for example by flushing the cellular materials from the primary medium(e.g., using physiological solutions, fixatives, etc.) directly into thesecondary medium (e.g., a recess, well, vial or other receptacle coupledwith or inserted into the support member or housing).

[0179] In one exemplary embodiment, cellular materials are collectedfrom expressed breast fluid onto a primary sample collection mediumcomprising a filter, particulate medium, or nitrocellulose membrane 39.When a nitrocellulose membrane is used, the cellular materials arewashed after primary collection from the membrane by a flushing rinse.For example, cytology fluid or another desired rinse liquid is used totransfer the cells from the primary collection medium into a secondarycollection medium, typically a fluid-retaining well or reservoirintegrated within, or coupled with, the support member 56 or the samplecollection housing 30.

[0180] In more detailed embodiments of the invention, a nitrocellulosemembrane 39 is employed for primary sample collection. The membrane istypically fluidly connected with the hand-held pump 10′ by seating ormounting the filter on or within a support member 56, as describedabove. Nitrocellulose membranes are particularly well suited forcollection of proteins, polynucleotides, or other soluble or suspendedconstituents of breast fluid. In preferred aspects, a nitrocellulosemembrane is selected and employed within the device for collection ofcytological specimens, particularly cells and cellular constituents. Asillustrated in FIGS. 16-18, the filter (optionally supported by asupport member) is positioned within the breast engaging member 14 nearthe base 22 of the engaging member proximate to the patient's nipplewhen the engaging member is seated against the breast. Comparing FIGS.16 and 17, activation of suction pressure by manual operation of thedevice draws the nipple 16 into closer proximity to, or into actualphysical contact with, the membrane, whereby the expressed fluid isefficiently transferred to the membrane.

[0181] Due to the fragility and fine porosity of nitrocellose membranes39, it may be necessary to employ additional measures to protect themembrane against negative vacuum pressure and contact with the nipple 16during operation of the pump 10′. In this regard, uniquely designedmembranes and filters are provided for use within the device whichfeature perforations or slits that disrupt the planar surface of themembrane or filter to facilitate air passage therethrough and impartstructural flexibility against mechanical perturbation. In one example,radial slits 280 emanate from a central disc portion 282 of the membraneor filter (FIG. 24) to allow passage of air through the membrane orfilter during vacuum pressurization and to increase structuralflexibility of the membrane or filter. Alternatively, one or more spiralperforations 284 (FIG. 25) or transverse slits 286 (FIG. 26) may be cutor stamped in the membrane or filter to achieve similar improvements interms of permeability and flexibility.

[0182] Where the target constituent for collection from the expressedfluid is whole cells or cellular components, these materials may bepartitioned onto the surface of a membrane or filter, typically anitrocellulose membrane 39, for further processing and cytologicalexamination. For this purpose, nitrocellulose membranes are employedwhich have a sufficiently small pore size to retain the cells orcellular debris on the outer (i.e., facing the nipple) membrane surface.For example, nitrocellulose membranes having a pore size of betweenabout 0.5 μ and 5.0 μ, preferably between about 1.0 μ and 2.0 μ, areuseful to partition whole cells on their surfaces.

[0183] As noted above, a fluid-retaining recess, well or reservoir maybe fluidly connected to either the support member 56 or the samplecollection housing 30 of the handheld pump device 10′ for primary and/orsecondary sample collection. In certain embodiments, the fluid-retainingreservoir comprises an integral, defined compartment or enclosure withinthe sample collection housing for receipt of breast fluid and/orconstituent samples thereof, including cytology specimens that may bewashed into the reservoir after primary collection, as described above.Alternatively, the fluid well or reservoir can be a separable componentof the sample collection housing, e.g., in the form of a flexible lineror rigid fluid reservoir member of the housing removably connected witha complementary housing member that partially or completely encloses orotherwise engages the fluid reservoir member.

[0184] Thus, as depicted in FIGS. 16 and 18, certain embodiments of theinvention employ a removable fluid reservoir member 288 of the housing30 for secondary sample collection of breast fluid components, includingcytology specimens. In preferred embodiments, the removable reservoirmember is provided in the form of a rigid sample collection tube orvial, exemplified by a standard cytology vial (i.e., a container havingapproximately the same general shape and dimensions as a standardcytology vial). The tube or vial is removably connected with acomplementary housing member, for example an outer casing member 290 ofthe housing that partially or completely encloses the vial. Preferably,the tube or vial is sealably coupled with the outer casing member, forexample by partially or completely nesting the vial within the outercasing member to form an airtight coupling therewith.

[0185] In various specific embodiments, the tube or vial engages aninner wall 292 of the casing member 290 and forms a generally airtightseal against it. For example, the casing member and vial may becomplementarily sized and dimensioned to provide substantially airtightcontact between the inner wall of the casing member wall and an outerwall 294, or a top end 296 or bottom end 298, of the vial when thecasing member and vial are coupled to form the assembled housing. Incertain embodiments, the outer wall of the vial features acircumferential ridge or fin 299 that engages and thereby makes acircumferential airtight seal against the inner wall of the casingmember when the vial is nested with the casing member (see, e.g., FIGS.16-20). In more detailed embodiments, the fin or ridge is replaced by aflexible O-ring 300 that seats in a circumferential O-ring groove 302 onthe outer surface of the vial and forms a circumferential seal with theinner casing wall.

[0186] The purpose of the sealable coupling between the outer casing 290and removable fluid reservoir 288 members of the housing 30 is to directvacuum pressure from the vacuum pump 160 to the breast engaging member14 in a path that includes the fluid reservoir member of the housing 30.In this manner, the removable reservoir is both gaseously and fluidlyconnected with the engaging member to facilitate secondary samplecollection. To accomplish these objectives, the removable reservoirmember is modified to include one or more air ports 310 that form agaseous connection between the lumen of the reservoir (which is in turnconnected to the engaging member, optionally via air channels 80 througha support member 56) and the vacuum pump. This allows the reservoirmember to remain in place during primary and/or secondary samplecollection, to function as both a conduit for vacuum pressuretransmission to the breast and a receptacle for fluid sample materials(e.g., to directly collect expressed fluid or as a secondary collectionmedium to receive primarily collected sample materials washed orotherwise transferred from the primary sample collection medium).

[0187] In the latter context, the removable reservoir member 288 of thehousing 30 may communicate for both fluid and gaseous transmissiondirectly with the breast engaging member 14 of the device, or indirectlyby way of the air channels 80 in the support member 56 optionallycoupled with the engaging member. As noted above, the air channels canserve a dual purpose as channels for vacuum pressure transmission aswell as transfer of fluids from the primary sample collection medium,through the air channels, into the removable reservoir (as exemplifiedby transfer of “wash” fluid containing cells and cellular componentsflushed from a primary collector, e.g., a nitrocellulose membrane,mounted in the support member, through the air channels, and into thesecondary collection fluid reservoir). To facilitate this and relatedpurposes, the channels may extend through tubular basal columns 311 orother fluid connection ports that extend from the support member toward,or into, a lumen 314 of the reservoir member of the housing.

[0188] In the embodiment of a hand-held pump device 10′ illustrated inFIGS. 1620, the reservoir member 288 is a cytology vial removably,sealably coupled with the outer casing member 290 to form the assembledhousing 30. To achieve this sealable coupling, the outer wall 294 of thevial features a circumferential fin 299, or a flexible O-ring 300 seatedin a circumferential O-ring groove 302, that forms a circumferentialseal with the inner wall 292 of the casing member (see, e.g., FIGS. 16,19 and 20). The vial incorporates one or more air ports 310 thatcommunicate between the outer wall and the inner lumen 314 of the vialto form a gaseous connection between the lumen of the vial, the vacuumpump 160, and the breast engaging member 14. Preferably, multiple airports are provided, which are located on the side of the vial positionedbelow (i.e., toward the bottom end 298 of the vial) the ridge or O-ringthat forms the gaseous seal with the wall of the casing member.Alternatively, the air ports can be located at other positions on thevial, e.g., in a lower side wall or floor wall 318, provided theposition is suitable to maintaining the desired path of vacuum pressureflow and retaining fluids within the reservoir. In this context, it isnoted that certain embodiments of the fluid reservoir member of thehousing will dictate changes in the position of the air port(s). Forexample, where a cytology vial-shaped reservoir is selected, as shown inFIGS. 16 and 20, the floor wall may be raised relative to the bottom end298 of the vial, to reduce the sample volume of the vial for handlingand processing purposes. To accommodate this and other designs, the airports are desirably positioned in the outer (side) wall 294 of thereservoir between the floor wall and the sealing flange 299 or O-ring300.

[0189] Referring to FIGS. 19 and 20, further modifications of theremovable fluid reservoir member 288 of the housing 30 provides formulti-purpose use of the reservoir member for sample collection in aclinical setting, as well as sample storage, transport and/or processingin a laboratory setting. To facilitate these latter purposes, theremovable reservoir member may be provided with closure means forclosure of the reservoir after sample collection is completed—to preventsample contamination and spillage. Taking the cytology vial reservoirfor example, the top end 296 of the vial may be adapted to provide asealable primary closure for the main opening of the vial. For example,the vial top end can be provided with complementary threads 320 or otherclosure means to receive a conventional cap that is sized anddimensioned (e.g., complementarily threaded) to sealably engage the vialtop end. In conjunction with this use, and further considering the noveladaptation of the fluid reservoir member to provide a secondary opening(i.e., the integral air port 310 that functions as a vacuum connection),the reservoir is also equipped with secondary closure means to sealablyclose the air ports after sample collection. A variety of air portconfigurations are contemplated, which can be sealed using acommensurate variety of closure mechanisms. For example, the air port orports can be sealed using a flexible (e.g., rubber) stopper shaped anddimensioned to sealably plug into the port opening. Alternatively, theport can be closed by an adhesive seal or sticker that adheres to theouter wall 294 of the reservoir member surrounding the port opening.

[0190] With respect to the latter secondary closure design, theinvention provides a combined closure and labeling device 324 whichfunctions both as a secondary closure mechanism to seal the air port 310of the removable reservoir and as a labeling template to provide aconvenient writing surface for sample labeling (see, e.g., FIGS. 21 and22). This aspect of the invention may be achieved, for example, byproviding any of a variety of adhesive closure/labeling tabs that may bedirectly applied to sealed air port after sample collection. This simpletype of closure/labeling tab can, for example, be provided as a separateadhesive sticker having a first, closure-forming surface 325 bearing anadhesive coating 326 on at least a portion of said surface, forapplication over the air port to form a seal, e.g., by adhesive contactwith the outer wall 294 of the removable reservoir. The adhesive coatingpreferably forms a seal that is resistant to disruption by contact withaqueous solutions and other materials present in the collected samples,for example buffers and fixatives. The adhesive coating is alsopreferably shielded before use by a protective tab or other protectivesurface 327 that covers the coating and is removed therefrom prior touse to expose the adhesive coating.

[0191] The closure/labeling device 324 has a second, labeling surface328 opposite the closure-forming surface 325 that is made of a blanktemplate material suitable for receiving a stable, ink or graphiteimprint thereon. For example, the second surface may be made ofcellulose or other fibrous material adapted for imprinting sample dataupon the surface thereof using a pen, pencil or other writing implement.Alternatively, the second, labeling surface may be made of plastic orother material adapted for retaining data imprinted in permanent ink(e.g., using an alcohol-based marker). When the adhesive tab is appliedover the air port, the blank template material of the second, labelingsurface of the tab is facing outward and at least a portion of thesurface covers a smooth portion of the outer wall of the removablereservoir 288, to allow the doctor or technician to imprint a clear datarecord on the labeling surface.

[0192] In more detailed embodiments, the closure/labeling device 324 isaffixed to the removable reservoir 288 during operation of the hand-heldbreast pump 10′ in a first, open configuration that leaves the air port310 uncovered for transmission of vacuum pressure (see, e.g., FIG. 21).The closure/labeling device that is thus pre-attached to the reservoircan be manually repositioned, or otherwise manipulated, after samplecollection to a second, closed configuration to form a seal or closureagainst the air port (see, e.g, FIGS. 21-23). In the exemplaryembodiment shown in FIGS. 19-23, the closure/labeling device comprisesan adhesive strip that is folded in a first, open configuration (FIG.21) to form two layers-an inner layer 330 that is affixed to thereservoir proximate to, but not covering, the air port, and an outerlayer 332 that folds back over the inner layer in the openconfiguration. In this embodiment, the outer layer of the strip providesboth the first, closure-forming surface 325 with the adhesive coating326 for securing closure of the strip, and the second, labeling surface328 formed of the blank template material opposite the closure-formingsurface. In the open configuration, the outer layer is optionallysecured in the folded-back position against the inner layer byengagement of the labeling surface with the inner layer, for example bya second adhesive coating 336 on the inner layer that holds the twolayers together in the folded position. Also optionally, the adhesivecoating 326 of the first, closure-forming surface may be protected inthe open configuration by folding an end segment 338 of the outer layer325 bearing the adhesive coating 326 for closure back on itself, so thatthe closure forming surface 325 provides the protective surface 327 toshield the adhesive prior to closure (as shown in FIGS. 21 and 22). Tomanipulate this closure/labeling device into the second, closedconfiguration, the end segment can then be lifted and pulled outward torelease the outer layer 332 from the inner layer 330 and to unfold theend segment to separate the adhesive coating 326 on the closure-formingsurface 325 from the protective surface 327. The outer layer 332 is thenunfolded away from the inner layer and wrapped around the reservoir sothat the closure-forming surface covers the air port to form a closurethat is water-tight or water-resistant to effectively prevent samplespillage from the reservoir and contamination. In this context, closureis effectuated by direct apposition of the adhesive coating 326 to theouter wall 294 of the reservoir surrounding the air port, or by drawingthe outer layer tight across the air port and annealing the adhesivesurface back upon the inner layer. In either case, manipulation of theclosure/labeling strip to the closed configuration positions thelabeling surface 328 to face outward for easy recordation of sampledata. In yet additional detailed embodiments, the closure/labeling stripcan be better secured against dislodgement during loading and removal ofthe reservoir 288 by including one or more circumferential guide ridges340 (see FIGS. 19 and 20) on the exterior of the reservoir, to shieldthe closure/labeling strip from mechanical dislodgement and/or toincrease the fidelity of the closure against leakage.

[0193] In related aspects of the invention, a novel breast fluidcollection reservoir is provided for use within a mammary fluidcollection device of the invention, which incorporates the foregoingfeatures of the removable reservoir member of the sample collectionhousing. The novel collection reservoir as described in the foregoingpassages is useful within the breast fluid collection methods of theinvention, as well as within various sample handling, processing, anddiagnostic assay methods performed in the laboratory subsequent tocollection of a breast fluid sample.

[0194] The novel breast fluid collection reservoir of the invention istypically provided in the form of a rigid tube or vial, for example inthe form of a modified cytology vial, having a top end which defines aprimary opening for access to the sample. The reservoir furthercomprises an outer reservoir wall, typically a cylindrical sidewall of atubular reservoir closed at a bottom end thereof, e.g., by a floor wall,as described above. The outer reservoir wall defines one or more airports that communicate between the outer wall and an inner lumen of thevial. In more detailed embodiments, the fluid-retaining reservoircomprises a removable fluid reservoir member of a sample collectionhousing of a mammary fluid collection device. Typically, reservoirmember is a rigid sample collection tube or vial removably connectedwith an outer casing member of the sample collection housing of thecollection device.

[0195] Within this aspect of the invention, the reservoir may be adaptedfor removable, sealable connection with the outer casing member of saidhousing, to form an airtight coupling therewith. In certain embodiments,the fluid-retaining reservoir is a cytology vial sealably connectablewith the outer casing member to form the airtight coupling. For example,the fluid-retaining reservoir can be removably nested within the casingmember to form a substantially airtight contact between an inner wall ofthe casing member wall and an outer wall, or a top or bottom end, of thereservoir member. To achieve this function, the outer wall of thefluid-retaining reservoir may be provided with a circumferential ridge,fin or O-ring adapted to engage and make a circumferential airtight sealagainst the inner wall of the casing member.

[0196] In more detailed aspects, the fluid-containing reservoir memberfor use within the devices and methods of the invention includes aclosure device for closing the reservoir after the sample of mammaryfluid is introduced therein, to prevent sample contamination andspillage. The closure may comprise a simple cap adapted to sealablyengage a top end of the reservoir. The cap or similar “primary closure”may extend to cover the air port(s) of the reservoir, which may becontiguous with the top opening of the reservoir or separate therefrom.In alternative embodiments, secondary closure means may be providedwhich are specifically adapted for closure of the air port(s). Thesecondary closure means may comprise a plug, cap or adhesive seal orsticker sized and constructed to engage or adhere to the outer wall ofthe reservoir member at or surrounding the air port opening(s) to formthe closure.

[0197] In certain embodiments, the secondary closure means comprises acombined closure and labeling device which functions as a secondaryclosure mechanism to seal the air port(s) of the reservoir and as alabeling template to provide a writing surface for sample labeling.Often, the secondary closure means comprise a combined closure andlabeling tab or sticker for application to the outer wall of thereservoir to seal the air port after the sample is collected. The tab orsticker has a first, closure-forming surface for application over theair port to form a seal by juxtaposition or adhesive contact with theouter wall of the reservoir, and a second, labeling surface opposite theclosure-forming surface made of a blank template material for imprintingwritten information thereon. The first, closure-forming surfacetypically has a water-insoluble adhesive coating on at least a portionof the surface. In more detailed embodiments, the tab or sticker ispre-attached to the removable reservoir member in a first, openconfiguration and can be manually repositioned or otherwise manipulatedafter sample collection to a second, closed configuration to form a sealor closure against the air port(s), as described in detail above.

[0198] In yet additional, related aspects of the invention, methods forbreast fluid sample collection, sample handling, and/or sampleprocessing are provided which incorporate the novel fluid-retainingreservoir adapted for use with a hand-held breast pump 10′ of theinvention. These methods include, generally methods for collectingbreast fluid samples which involve collecting expressed mammary fluid ina modified fluid reservoir as described above. Additional methodsinvolve loading and removal of a modified fluid-retaining reservoirwithin a hand-held breast pump, according to the above description.Related methods include an additional step of securing the primaryand/or secondary closure means of the reservoir after a sample ofmammary fluid, or a component thereof, is collected therein.

[0199] Related to these methods, the invention provides additionalmethods for handling or processing biological samples of mammary fluid,or components thereof, for use in a diagnostic assay to detect orquantify a breast disease marker in the sample. The methods generallyinvolve providing or obtaining the biological sample of mammary fluid ora mammary fluid component in a specialized fluid-retaining reservoiraccording to the above description. The sample may be initiallycollected by oxytocin induction or by application of a breast pump 10,10′ of the invention without oxytocin priming. The reservoir istypically provided as a flask, vial, or tube that has a top end defininga primary opening for collection of, and later access to, the mammaryfluid sample, and an outer reservoir wall that defines one or more airports communicating between the outer wall and an inner lumen of thevial.

[0200] In more detailed handling and processing methods, the reservoirincorporates specialized closure means, for example a cap that securesthe top end of the reservoir and secondary closure means to sealablyclose one or more air port(s) of the reservoir, to close the reservoirafter the sample is collected and thereby prevent sample contaminationand spillage. Typically, the fluid-retaining reservoir is a modifiedcytology vial adapted as a removable reservoir member integrated with ahand-held mammary fluid collection pump 10′ as described above. Moredetailed handling/processing methods employing the novel reservoirinclude the step of accessing said sample within the reservoir totransfer or process the sample for detection or quantification of abreast disease marker. Additional methods include one or more steps ofprocessing the sample to detect or quantify the breast disease marker.

[0201] In additional detailed embodiments, the step of processing thesample for marker detection comprises fixing or staining cells or cellfragments in the sample, before or after transfer of the sample from thereservoir, e.g., for cytological analysis. Yet additional methodsinvolve exposing the sample in the reservoir or after transfer to aprocessing reagent, e.g., a fixative, labeling reagent, buffer, etc., toprepare sample components, including whole mammary fluid, whole cells,cell fragments, cell membranes, purified proteins, bulk proteins,glycoproteins, peptides and/or polynucleotide components, for furtherprocessing, which may include detection or quantification as theselected breast disease marker(s).

[0202] As noted above, certain modular designs for the hand-held breastpump 10′ of the invention feature a separate breast engaging member 14provided as a funnel shaped component adapted for removable coupling toone or more interconnecting components of the device for cleaning andinterchanging of parts. Typically, the breast engaging member isremovably coupled with the sample collection housing 30. In oneembodiment shown in FIGS. 16-18, the housing is comprised of multiplemembers, exemplified by an outer casing member 290 and a removable,fluid reservoir member 288. As illustrated in FIG. 18, the engagingmember may be directly coupled to the fluid reservoir member, which isin turn engaged by a sealable connection (e.g., a sealable compressionfit) with the outer casing member of the housing as described above. Inthis context, one alternative coupling design is for the engaging memberto be fitted with mounting threads 340 or other coupling means to couplewith complementary threads 320 or other closure means on the top end 296of the removable reservoir (e.g., cytology vial). In this manner, theclosure means of the removable reservoir, adapted to receive aconventional cap that sealably engages the reservoir top end, serves thedual purpose of coupling the engaging member 14 with the remainingmodular components of the device. Alternative coupling means are ofcourse contemplated as well, as exemplified by a simple pressure fitcoupling to removably couple mated ends of the engaging member andreservoir member. To facilitate stable coupling of the engaging memberwith the sample collection housing 30, the housing may be furtherelaborated to include flared extensions 350 terminating in outwardlyreflected feet 352 collectively shaped and dimensioned to engage aninner, circumferential groove 354 underlying the flared or reflectedterminal edge 18 of the engaging member (see, e.g., FIG. 18).

[0203] In addition to the foregoing features, the hand-held breast pump10′ of the invention may optionally include any of the alternativefeatures described above for the general-purpose breast pump 10,including different solid phase sample collection media and supportmember 56 designs. Thus, the support member for use with the hand-heldpump can also include a fluid-retaining well which may be optionallyfilled with a desired solution, such as a buffer, a solution containinga probe, cross-linking agent, blocking agent, denaturing agent, etc., tofacilitate sample collection, handling, and/or processing. Alternativedesigns and configurations of the housing 30 and/or support member 56are also provided which vary with the type of solid phase samplecollection medium used. For example, when a particulate solid phasesample collection medium 41 (e.g. beads, resins, or microspheres) isused, the medium may be enclosed in a cartridge 82 removably mounted to,or integrated within, the support member or otherwise removablyconnected to the sample collection housing 30, as described in detailabove. It is also contemplated to adjustably mount the solid phasemedium relative to the housing 30 of the hand held pump device, so thatthe collection medium can be moved closer to, or farther away from, thebase 22 of the engaging portion 14 of the pump 10. In this regard,various designs are contemplated commensurate with the above descriptionto provide a reciprocating mechanism which adjustably moves the solidphase collection medium in closer, or more distant, proximity to thenipple when the hand-held breast pump is engaged therewith.

[0204] Within more detailed aspects of the invention, the hand-heldbreast pump 10′ typically incorporates a compact vacuum pump housing 410which structurally and functionally integrates the vacuum pump 160 withthe sample collection housing 30 and, in turn, with the engaging member14 (see, e.g., FIGS. 16-18). The vacuum pump housing is in turn coupledwith, or is modified to include, a vacuum pump actuating mechanism. Theactuating mechanism may be in the form of a switch, button, lever orother actuation device suitable for use with the selected vacuum pump.As noted above, a variety of vacuum pumps may be incorporated within thebreast pump device, including any manual or electric, piston, hydraulicor diaphragm pump of suitable size and dimension for incorporation inthe handheld pump vacuum housing. In the exemplary embodiment depictedin FIGS. 16-18, the vacuum pump is a conventional diaphragm pump, andthe pump actuation mechanism is a simple hand lever 420 pivotallyconnected to the pump housing or other suitable connection point.

[0205] Exemplifying this aspect of the invention, the pump housing isoptionally coupled with, or extended to include, an opposing handle 422to facilitate depression of the hand lever by gripping and manualclosure of the lever toward the handle (compare FIGS. 16 and 17). Thehandle is preferably molded or cast as an integral extension of thevacuum pump housing. In more detailed embodiments, exemplified in FIGS.16-18, the vacuum pump housing and handle are molded or cast together asan integral unit or modular component with the outer casing member 290of the sample collection housing 30. The handle defines a pivot recess430 or detent that pivotally receives a pivot head 432 or shaft joinedor integrated with the pump actuation lever (as shown in FIGS. 16 and17).

[0206] In this manner the pump actuating lever 420 is pivotallyconnected to the pump housing 410 or handle 422 by a pivotal connection430, 432 that joins the actuation lever and pump housing in an easilyassembled fashion, e.g., by snap fitting the pivot head 432 of the leverinto the pivot recess 432 of the handle. The actuation lever easily andeffectively actuates the vacuum pump 160 by depressing the lever to drawa flexible diaphragm member 440 downward, away from a primary vacuumchamber 442 connected with, or integrated within, the sample collectionhousing. In the embodiment shown in FIGS. 16-18, the primary vacuumchamber is integrally formed as a channel within the vacuum pump housingproximate the flexible diaphragm member and extending to a communicatingport 444 opening to the inner wall 292 of the outer casing member 290 ofthe housing. The remainder of the vacuum path (i.e., through the outercasing member to the air port 310 of the removable fluid reservoirmember 288 of the housing into the lumen 314 of the reservoir,optionally through air channels 80 of the support member 56, and to thebreast engaging member) is described above.

[0207] To engage and move the flexible diaphragm member 440 in thisfashion, the actuation lever engages a diaphragm retraction mechanism,for example comprising a reciprocating shaft 450 or piston sealablyconnected through the diaphragm to a diaphragm-engaging head 452, tocollectively translate depression movement of the lever to downwardretraction of the flexible diaphragm member (compare, e.g., FIGS. 16 and17). These components of a diaphragm pump are conveniently housed withina protective pump cover 460 which engages a complementary rim 462 orother coupling surface of the pump housing 410 by a complementaryengagement fitting (arrows 464). This coupling may optionally serve tosealably anchor the flexible diaphragm member, e.g., by sandwiching aperipheral edge 466 of the diaphragm between the cap and housing asshown in the figures. Optional pump devices, actuation mechanisms, andpump housing designs are contemplated within the invention, which arewithin the level of skill in the art to engineer for use with thehandheld breast pump devices disclosed herein.

[0208] As noted above, mammary fluid expression and collection using thedevices of the invention may be facilitated in certain instances byprior or concurrent administration of the peptide hormone oxytocin, oran analog thereof, in an amount that is effective to stimulatemyoeptithelial contraction in the alveolar gland ducts of the breast tofacilitate expression of the mammary fluid from the nipple. Preferablythe oxytocin preparation is administered intranasally and isadministered in an amount that is intranasally effective to stimulateexpression of mammary fluid from the nipple. Alternatively, anintramuscular or intravascular injection of oxytocin can effect the samemyoepithelial contraction response as the intranasal administrationroute. The amount, timing and/or mode of oxytocin administration may beadjusted on an individual basis depending on such factors as menstrualcycle stage, use of birth control or hormone replacement therapy,pregnancy history, age of onset of menarche, ethnicity and other factorsknown to affect an individual's propensity for breast fluid expression.

[0209] Oxytocin is a peptide hormone of pituitary origin that isnaturally released into the bloodstream of lactating women in responseto suckling, and stimulates contraction of myoepithelial cells in themammary alveoli and ducts to cause milk ejection (Cobo, J. Perinat. Med.21:77-85, 1993). The drug has also been widely used for stimulatinglabor in pregnant women, due to its activity of stimulating uterinecontractions (Satinet al., Am. J. Obstet. Gynecol. 166:1260-1261, 1992).For these reasons, the pharmacology of oxytocin has been thoroughlyinvestigated, including detailed studies of effective dosages, half-lifeand potential side effects.

[0210] For use in the present invention, an oxytocin preparation isprovided for intranasal, intramuscular, or intravenous administrationthat contains oxytocin in a biologically suitable, liquid carrier. Asused herein, “oxytocin” refers to natural or synthetic oxytocin andbiologically active derivatives and analogs thereof. Naturally occurringoxytocin from mammalian sources is of course suitable, as are otherknown, naturally occurring oxytocin-like peptide analogues and theirsynthetic counterparts having similar activities for stimulatingalveolar-ductal myoepithelial contraction. Preferably, the oxytocin usedwithin the invention is a simple peptide hormone comprising a cyclicpeptide, the peptide having a well defined ring portion(Cys-Tyr-Ile-Gln-Asn-Cys) and tail portion (Pro-Leu-Gly). However,numerous derivatives and analogues are known, or readily obtainable, inthe art, e.g., derivatives or analogues having amino acid truncations,deletions or substitutions at one or more residues of the peptide andwhich exhibit substantially the same activity as naturally occurringoxytocin (i.e., having at least 75%, and preferably 85%-95% or more,activity compared to that of native oxytocin for stimulatingalveolar-ductal myoepithelial contraction). The most economic oxytocinpreparations for use within the invention contain a synthetic oxytocin(e.g. Pitocin® or Syntocinon® available from various providers, forexample Sandoz (Basel, Switzerland) and United States Pharmacopeia.Alternate benefits may be obtained with the use of a long-actingoxytocin analog within the methods of the invention. The utility andpharmacokinetics of such analogs, exemplified by the peptide analogcarbetocin, are described in detail in U.S. patent application Ser. No.09/481,058 filed Jan. 11, 2000 (incorporated herein by reference).

[0211] For use with the methods, devices and kits of the invention, apreferred oxytocin preparation contains approximately 40 USP units ofoxytocin per ml of liquid carrier. Preferred liquid carriers arebiologically compatible solutions, such as a lactated Ringer's solutionor other physiologically balanced, sterile, non-toxic and non-irritativesolution. To administer the oxytocin intranasally, a standard nasalsqueeze bottle is used, which delivers approximately 0.5 ml of theoxytocin preparation into the patient's nostril when squeezed. Theoxytocin is absorbed by the nasal mucosa into the systemic circulationwhere it reaches and acts specifically on the myoepithelial cellssurrounding the alveoli of the breast and making up the walls of thelactiferous ducts, causing their smooth muscle fibers to contract andforce any fluids present into the large ducts or sinuses where it can beexpressed from the nipple spontaneously onto a sample collector or bythe further action of a breast pump. Intranasal application of the spraypreparation is therefore a practical and effective method ofadministration. The half-life of oxytocin in the human bloodstream isextremely short, estimated to be about 10-15 minutes or less, due to itsrapid removal from plasma by the kidney, liver, and mammary gland, andthe time to pharmacokinetic and clinical steady state is readilydetermined depending on the mode of administration (e.g. bolus dosage,repeat administration, or steady infusion). (See for example, Gonser,Arch. Gynecol. Obstet. 256:63-66, 1995; and Orhue, Obstet. Gynecol.83:229-233, 1994, each incorporated herein by reference in itsentirety). It is therefore a routine matter to determine an appropriateconcentration and dose of the oxytocin preparation to administer aneffective amount (either intranasally effective, intravenouslyeffective, or intramuscularly effective) of the oxytocin to causeexpression of mammary fluid with or without the assistance of a breastpump. (See for example, Newton, Ann. N.Y. Acad. Sci. 652:481-483; Mena,Neuroendocrinology 61:722-730, 1995; Gonser, Arch. Gynecol. Obstet.256:63-66, 1995; Orhue, Obstet. Gynecol. 83:229-233, 1994; Satin et al.,Am. J. Obstet. Gynecol. 166:1260-1261, 1992; and Satin et al., Obstet.Gynecol. 83:234-238, 1994, each incorporated herein by reference in itsentirety).

[0212] Although not all female patients are expected to be responsive tointranasal oxytocin stimulation, an intranasally effective amount ofoxytocin for the purposes of the invention can be readily determined. Asused herein, an intranasally effective amount of oxytocin is an amountof oxytocin sufficient to intranasally stimulate the expression of atleast 3 μl of mammary fluid in at least 50% of non-lactating femalepatients with the aid of negative pressure to the nipple of between50-200 mm Hg applied by the breast pump (up to 45 min after a firstadministration of the oxytocin spray). It may be necessary, and indeedpreferred, to administer a low, preliminary dose of oxytocin to thepatient, for example a single spray of a 40 Unit/ml oxytocin solution ineach nostril, or multiple sprays of a lower concentration oxytocinpreparation, and thereafter wait to determine a particular patient'ssensitivity. If there is no reaction with an initial application of thebreast pump after a short post-administration period of 2-15 minutes,and preferably 2-5 minutes, a booster dose of the oxytocin spray may beadministered and the pump reapplied. In this way, the clinician canmodulate the dosage to each patient's varying sensitivity, and therebyminimize potential adverse side effects. Alternatively, an effectivedose of intramuscular or intravenous oxytocin can be used according tothe same dosage determination and administration principles in patientswhere intranasal administration fails or is otherwise contra-indicatedas a preferred mode of administration.

[0213] As noted above, the amount, timing and/or mode of oxytocinadministration may be adjusted based on specific factors known to renderindividuals more or less sensitive to induction of breast fluidexpression. These factors are generally well known in the art, andinclude, for example, menstrual cycle stage, use of birth control orhormone replacement therapy, pregnancy history, age of onset ofmenarche, and ethnicity, among other factors.

[0214] Thus, in one aspect of the invention, methods for obtaining abiological sample from a patient and/or determining the amount of abreast disease marker in a biological sample from breast fluid areprovided which include a step of determining a menstrual stage of thepatient. Based on the determined menstrual stage, a drug administrationprotocol is selected having a predetermined oxytocin dosage, timingand/or frequency of oxytocin delivery, and/or mode of oxytocinadministration.

[0215] According to these methods, one or more variables of oxytocindosage, timing and/or frequency of oxytocin delivery, and/or mode ofoxytocin administration are selected depending on whether the patient isstaged within one of five approximate menstrual phases. These phasesinclude 1) a proliferative phase (characterized by a tight configurationof the alveolar lumena); 2) a follicular phase (characterized by adefined configuration of the alveolar lumena); 3) a luteal phase(characterized by an open configuration of the alveolar lumena, withsome secretion by the alveolar cells); 4) a secretory phase(characterized by an open configuration of the alveolar lumena, withsecretion by the alveolar cells); and 5) a menstrual phase(characterized by a distended configuration of the alveolar lumena, withsecretion by the alveolar cells).

[0216] It is generally not desired to conduct the methods of theinvention for patients staged in the proliferative or follicular stageof their menstrual cycle (approximately 3-7 days and 8-14 days,respectively). However, in some circumstances sample collection can beperformed for these individuals using high and/or repetitive doses ofoxytocin and otherwise optimizing the breast fluid expression responseby selecting a particular mode of oxytocin administration, orcombination thereof (e.g., intravenous administration followed byintranasal administration). For patients staged in the luteal orsecretory stage of their menstrual cycle (approximately 15-20 days and21-27 days, respectively), intermediate dosages of oxytocin are selectedand repetitive administrations are reduced or eliminated. For patientsstaged in the menstrual phase, dosages of oxytocin and repetitiveadministrations are reduced even further while still providing aneffective administration protocol to yield sufficient breast fluidexpression.

[0217] Determination of effective administration protocols for patientsof different menstrual stages can also be readily achieved within theinvention. As used herein, an effective administration protocol yieldsat least 3 μl of expressed mammary fluid in at least 50% ofnon-lactating female patients at an equivalent menstrual stage with theaid of negative pressure to the nipple of between 50-200 mm Hg appliedby a breast pump up to 45 min after a first administration of theoxytocin spray. Various combinations of oxytocin dosage, timing and/orfrequency of oxytocin delivery, and/or mode of oxytocin administrationare contemplated, as can be readily determined by the skilled artisan inaccordance with the teachings herein. Likewise, it will often bepreferred to administer a low, preliminary dose of oxytocin to thepatient and thereafter wait to determine a particular patient'ssensitivity, even when an individual's menstrual stage has beendetermined and a particular administration protocol selected. Thus, ifthere is no reaction with an initial application of the breast pumpafter a short post-administration period, a booster dose of the oxytocinmay be administered and the pump reapplied. In this way also, theclinician can apply a first, stage specific dose of oxytocin andthereafter modulate the dosage, period of time between boosteradministrations, and/or mode of administration, to each patient'svarying sensitivity.

[0218] In other, related aspects of the invention, methods for obtaininga biological sample from a patient and/or determining the amount of abreast disease marker in a biological sample from breast fluid areprovided which include a step of determining a non-menstrual stagepatient sensitivity index. Examples of such indices include 1) patientuse of hormone based birth control; 2) patient use of hormonereplacement therapy; 3) patient pregnancy history; 4) patient age ofonset of menarche; and 5) patient ethnicity. Other indices associatedwith sensitivity to induction of breast fluid expression are alsocontemplated. These factors can be determined by such routine steps aspatient consultation, evaluation of patient records, and clinical orlaboratory-based analysis (e.g., physical screening, measurement ofsex-steroid hormone levels, etc.) Based on a determined non-menstrualstage sensitivity index, an effective drug administration protocol isselected having a predetermined oxytocin dosage, timing and/or frequencyof oxytocin delivery, and/or mode of oxytocin administration, inaccordance with the methods described above. In yet additional methodsan effective drug administration protocol is selected by firstdetermining both a patient's menstrual stage and at least onenon-menstrual stage sensitivity index specific to the patient, andthereafter selecting an effective oxytocin administration protocol basedon these combined indices.

[0219] In yet additional methods within the invention, it may bepreferred to conduct the foregoing sample collection methods inconjunction with a conventional mammographic procedure. In this manner,costs, time and patient discomfort can be minimized. Further, byconducting the sample collection immediately following a mammogram it isexpected that breast fluid expression may be facilitated by breastmanipulation during the initial procedure. Additional steps tofacilitate breast fluid expression include manual breast massage andapplication of heat packs to the breast.

[0220] For mammary fluid collection using a breast pump 10, 10′ of theinvention, alone or in conjunction with oxytocin stimulation, the breastpump is applied and negative pressure is generated on the breast tofacilitate the expression of mammary fluid. Within the methods of theinvention, negative pressures of 50-200 mm Hg are preferred, and thesepressures are maintained, preferably intermittently, for approximately1-15 minutes, depending on the sensitivity of individual patients,oxytocin dosage and other factors. The volume of expressed mammary fluidwill vary depending on a variety of factors, including the time andpressure of breast pump administration, and other factors. For the leastsensitive breast marker assays of the invention, a volume of expressedmammary fluid of 300-500 μl is preferred to provide ample material forconducting the assay, and these volumes will be obtainable from asubstantial proportion of women treated according to the above methods.To express 300-500 μl of mammary fluid, some women will require repeatedstimulation treatments, perhaps requiring pooling of mammary fluidsamples obtained during multiple patient visits. However, for moresensitive assays of the invention, e.g. solid phase immunoassays, muchsmaller samples of 3 μl or less will be suitable to carry out theassays, particularly in the case of breast cancer markers that arenaturally secreted into the mammary fluid and are therefore expected tobe present in very high concentrations compared to, for example, breastepithelial cell surface antigens or intracellular antigens that are notsecreted.

[0221] The following examples are offered by way of illustration, not byway of limitation.

EXAMPLE 1 Induction of Mammary Fluid Expression by Application of aNovel Breast Pump/Mammary Fluid Sample Collection Device

[0222] Within the present example, a hand-held breast pump device 10′ isemployed to collect a primary sample of mammary fluid componentscomprising whole cells and cell fragments for cytological examination.The doctor, technician or patient collects the breast fluid specimen bygrasping and operating the hand-held pump as described above tostimulate expression of the mammary fluid and collect a specimenthereof. This operation is a one-handed procedure, leaving the physicianor technician free to conduct additional activities with the other, freehand. In this regard, the compact hand-held pump design allows thedevice to be picked up and manipulated with one hand, to seat the breastengaging element against the breast, apply sufficient vacuum pressure tothe breast by manual operation of the vacuum pump to cause a suitablevolume of breast fluid to be expressed at or near the nipple, and tosimultaneously collect at least a primary sample of expressed breastfluid onto, or within, the solid phase sample collection medium (e.g., anitrocellulose filter) without additional manual steps or the need toremove the device from the breast or engage two hands in the operation.

[0223] In the present example, the hand-held pump device 10′ is employedto collect a primary sample of a selected mammary fluid componentcomprising cells and other cellular materials, that are retained (e.g.,by retaining cells on a nitrocellulose membrane 39 or filter 40 coupledwith the breast engaging member 14 or sample collection housing 30) forfurther processing. The primary collected sample of cells and othermaterials is then transferred (e.g., by washing or manual transfer) to areservoir or other solid phase template, for further storage, processingand/or analysis. In more specific protocols, whole cells are separatedfrom expressed mammary fluid onto a nitrocellulose membrane 39 or afilter 40 secured in fluid connection with the breast engaging member 14a removable support member 56 mounted to the engaging member or samplecollection housing 30.

[0224] The cells are subsequently transferred or washed in fluid (e.g.,cytology fluid) to a second solid phase sample collection medium (e.g.,a slide, well, tube or vial), which may also be connected to, orintegrated with, the breast engaging member 14 or sample collectionhousing 30 as described above. In one protocol, the breast engagingmember is removed from the breast and a jet of cytological fluid isdirected against the filter on which the cells are retained. Thisflushes the cells off of the filter into an awaiting reservoir,typically a removable fluid-retaining reservoir. In this regard,uniquely designed membranes and filters are provided for use within thisprotocol which feature perforations or slits that disrupt the planarsurface of the membrane or filter to facilitate air passagetherethrough, to impart structural flexibility against mechanicalperturbation, and to allow cells flushed from a first side (facing thebreast) of the filter to a second side (facing internally, e.g., towardthe sample collection housing, when the filter is mounted with thedevice) for secondary sample collection. For example, radial slits 280,spiral perforations 284 or transverse slits can be made in the filter toallow passage of air and for cells partitioned onto the first surface ofthe membrane to be flushed through the membrane into the removable,fluid-retaining reservoir 288 seated in the sample collection housing30, for storage, shipment, and/or further processing directed towardcytological examination of the secondary collected sample.

EXAMPLE 2 Cytology in Biological Samples From Mammary Fluid

[0225] This example describes the use of conventional cytologicaltechniques to identify and classify breast diseases from samplesobtained as described in Example 1. Following collection of the sample,e.g. in a fluid-retaining reservoir member, the sample may be furtherprocessed (e.g., by centrifugation wherein the reservoir member is inthe form of a modified cytology vial). Processed samples are thentransferred to the central region of a clean glass microscopic slide,and a cover slip is slid over the sample to spread it along the surfaceof the slide. The slide is allowed to air dry and then is fixed, forexample in absolute alcohol, and stained with standard cytologicalstains, such as methylene blue, hematoxyln and eosin, and other suitablestains.

[0226] The slides are then examined by light microscopy for evidence ofatypical growth of cells and clumps of cells, using well known methods,including those described in Diagnosis of Non-Palpable Breast Lesions:Ultrasonographically Controlled Fine-Needle Aspiration: Diagnostic andPrognostic Implications of Cytology by Jacqueline Mouriquand, S. KargerPub., July 1993; Breast: Guides to Clinical Aspiration Biopsy by TildeS. and Irwin K. Kline, Igaku-Shoin Medical Pub., May 1988; Cytopathologyof the Breast (Asop Theory and Practice of Cytopathology 5 by ShahlaMasood, American Society of Clinical Pathology, November 1995; FineNeedle Aspiration Cytology and Its Clinical Applications: Breast andLung by Philip S. Feldman, American Society of Clinical Pathology,November 1984; each incorporated herein by reference in its entirety.

EXAMPLE 3 Stimulation of Mammary Fluid Expression for Sample Collectionby Coordinate Administration of Intranasal Oxytocin in Conjunction withApplication of a Novel Breast Pump/Mammary Fluid Sample CollectionDevice

[0227] The foregoing sample collection protocol in Example 1, as well asother sample collection methods within the invention, may be practicedsolely by the use of a novel breast pump 10, 10′ of the invention.Alternatively, these sample collection procedures may be practiced inconjunction with the use of oxytocin or oxytocin analogs to facilitateor increase mammary fluid expression induced by operation of the breastpump. As incorporated within the invention, these methods involveapplication of the breast pump 10, 10′ to the breast, optionally coupledwith oxytocin administration in amounts effective to facilitate mammaryfluid expression in the patient. After the sample is collected, abioassay is conducted on the sample to determine the presence and/oramount of a selected breast disease marker, preferably a breast cancermarker or panel of breast cancer markers, in the sample.

[0228] Oxytocin nasal solution acts specifically on the myoepithelialelements surrounding the alveoli of the breast and making up the wallsof the lactiferous ducts, causing their smooth muscle fibers to contractand thus force any fluids present into the large ducts or sinuses whereit can be expressed from the nipple by the further action of a breastpump. The nasal spray is promptly absorbed by the nasal mucosa to enterthe systemic circulation. Intranasal application of the spraypreparation is a practical and effective method of administration.Half-life of oxytocin in the human circulation is extremely short,approximately 10-15 minutes, and oxytocin is then rapidly removed fromplasma by the kidney, liver, and mammary gland.

[0229] Because of the known effects of oxytocin to cause uterinecontractions, pregnant women should not be treated by the methodscontained herein unless the benefits of testing outweigh the risk ofinducing premature labor.

[0230] The oxytocin nasal solution contains a concentration of naturalor synthetic oxytocin, or a functional analog thereof such ascarbetocin, that is intranasally effective in a selected volume ofadministered spray to stimulate expression of mammary fluid from anipple of the patient when a breast pump is applied to the nipple toassist mammary fluid expression. In the present example, a preferredoxytocin preparation containing approximately 40 USP units of oxytocinper ml of lactated Ringer's solution is administered into the nose withthe squeeze bottle held in the upright position while the patient is ina sitting position. One or two sprays are administered into each nostrilfrom a standard nasal squeeze bottle, which delivers approximately 0.5ml of the oxytocin solution per spray in a fine mist when the bottle issqueezed. The number and volume of sprays administered, as well as theconcentration of oxytocin in the solution, can be adjusted according towell known pharmacokinetic principles (See for example, Newton, Ann.N.Y. Acad. Sci. 652:481-483; Mena, Neuroendocrinology 61:722-730, 1995;Gonser, Arch. Gynecol. Obstet.256:63-66, 1995; Orhue, Obstet. Gynecol.83:229-233, 1994; Satin et al., Am. J. Obstet. Gvnecol. 166:1260-1261,1992; and Satinet al., Obstet. Gynecol. 83:234-238, 1994, eachincorporated herein by reference in its entirety) to ensure that theamount of oxytocin administered to the patient corresponds to anintranasally effective amount to stimulate the expression of at least 3μl of mammary fluid in at least 50% of non-lactating female patientsupon activation of the breast pump. For example, adjustments may bedesired in the number of sprays delivered b the patient, and/or thetiming of spray delivery, so that the clinician can modulate the dosageto each patient's varying sensitivity, and thereby minimize potentialadverse side effects. In the present example, a preliminary dose of asingle spray of the 40 Unit/ml oxytocin solution is delivered into eachnostril of the patient, and the administering clinician waits for ashort post-administration period of 2-3 minutes. After this period thebreast pump is applied, and the clinician determines whether or not theamount of oxytocin delivered was sufficient to facilitate or increasebreast pump-induced expression of mammary fluid. If additional fluidexpression is desired at this stage a booster dose of 1 or 2 additionalsprays of the oxytocin solution can be administered in each nostril, andthe pump reapplied after a 5-10 minute post-booster administrationperiod.

[0231] After the intranasally effective dose of the oxytocin isadministered and the clinician has allowed a suitablepost-administration period to elapse for the oxytocin to reach andstimulate the target alveolar-ductal tissue, the breast pump is applied.Negative pressures of 50-200 mm Hg are applied in the area of the nippleand are maintained, intermittently or continuously, for approximately1-15 minutes, depending on the sensitivity of individual patients,oxytocin dosage and other factors. Alternatively, oxytocin can beadministered by intramuscular or intravascular routes by well knownmeans (Oxytocin Injection (synthetic), USP; Wyeth-Ayerst Laboratories)to effect the same response as intranasal administration.

EXAMPLE 4 Verification of Sample Quantity, Origin and Quality

[0232] Using either of the above methods of Example 1 or Example 3,volumes of at least 3 μl of expressed mammary fluid can be collected ina substantial population of non-lactating female patients. During orafter the mammary fluid expression step, a biological sample iscollected from the expressed mammary fluid as described above. Forexample, a nitrocellulose filter may be placed within the breast pump inline with a path of the expressed mammary fluid into the pump, so thatthe expressed fluid contacts the filter. Upon contact of the primarysample of expressed mammary fluid with the filter, cells, proteins andother desired components of the mammary fluid adhere to the filter toform a filter-bound or filter-F retained biological sample forsubsequent analysis. Other suitable biological samples, including wholemammary fluid samples, cytological samples of whole cells, membranes orother cellular components, and samples containing proteins,glycoproteins, peptides, nucleotides and other constituents of theprimary mammary fluid sample can be collected with appropriatemodifications of the above procedures, according to well knownprinciples and methods.

[0233] To ascertain that the sample of mammary fluid is of mammaryorigin and is not corrupted by likely contaminants, one or moreconstituents of normal mammary fluid are assayed for. In the presentexample, an enzyme that is ordinarily present in mammary fluid,lysozyme, is assayed in the mammary fluid sample to help confirm thatthe sample is of mammary origin. Lysozyme (muramidase) is an enzymewhich hydrolyzes beta 1,4-glycosidic linkages in the mucopolysaccharidecell wall of a variety of microorganisms, which activity can be readilydetected and quantified using a routine, inexpensive assay. In thepresent example, Lysozyme is measured in the primary mammary fluidsample using the Quantiplate Lysozyme Test kit (Kallestad, Chasta,Minn.). The assay employs the reagents and procedures provided by themanufacturer and specified in detail in the manufacturer's instructions,with the exception that a mammary fluid sample is substituted in placeof serum, urine or tears. Analysis of these results establishes that thesample contains lysozyme, which is a normal component of human serum,urine, saliva, tears, nasal secretions, vaginal secretions, seminalfluid, and mammary fluid.

[0234] More specific assays are used in place of the lysozyme assay, orto supplement lysozyme assay results, particularly where clinical datafor human patients are being gathered. Other mammary fluid markers forsample verification that are more specific than lysozyme can be readilyincorporated within the invention, based on published and generallyknown information. In one example, the presence of cathepsin D isassayed using the monoclonal antibodies and methods disclosed inVetvicka et al., Biochem. Mol. Biol. Int'l. 30:921-928, 1993,incorporated herein by reference in its entirety). In another example,one or more human mammary epithelial antigens (HME-Ags) corresponding toglycoprotein components of the human milk fat globulin (HMFG) proteinare detected in the primary mammary fluid sample, or in the biologicalsample that is used in the breast cancer marker assay, using specificantibody probes, as described by Rosneret al., Cancer Invest. 13:573582,1995; Ceriani et al., Proc. Natl. Acad. Sci. USA 74:582-586, 1982;Ceriani et al., Breast Cancer Res. Treat. 15:161-174, 1990, eachincorporated herein by reference in its entirety). In many cases, thesample verification assay can be incorporated within the breast cancermarker assay in a single procedure, for example as described below inExample 4, an assay for HME-Ags (wherein the HME-Ag findings areindicative of sample origin/quality, and also of the presence and/orquantity of a specific breast cancer marker in the sample). In anotherexample, sample verification is achieved through a combinatorial (i.e.multiple marker) immunoassay targeting various cytokeratins, which canbe detected as a panel of cytokeratins specifically expressed in mammarytissue sample. (See, Nagle, J., Histochem. Cytochem. 34:869-881, 1986,incorporated herein by reference in its entirety). One or more of thesecytokeratins (e.g. K5, K8, K18 and K19) can be simultaneously orindependently measured in the context of a breast cancer assay, and thelevel of expression of the subject cytokeratin(s) can yield informationconcerning the presence or status of breast cancer in a patient. (Seefor example, Focus, Harvard University News Office, Mar. 21, 1991, pp.2-3; and Lee, Proc. Natl. Acad. Sci. USA 88:1991, each incorporatedherein by reference in its entirety).

EXAMPLE 5 Immunoassay for Human Mammary Epithelial Antigens inBiological Samples From Mammary Fluid

[0235] Human mammary epithelial antigens (HME-Ags) are glycoproteincomponents of the human milkfat globule (HMFG) and of the membrane ofthe breast epithelial cell, which are released by breast tumors and notby normal breast tissue. (Ceriani et al., Proc. Natl. Acad. Sci. USA74:582-586, 1977, incorporated herein by reference in its entirety). Inthe present example, several HME-Ags, having molecular weights of 150,70, and 45 kilodaltons, are detected and measured using specificanti-HMFG or anti-human mammary epithelial (α-HME) probes prepared andemployed as described by Ceriani et al., Proc. Natl. Acad. Sci. USA79:5420-5425, 1982 (incorporated herein by reference in its entirety).

[0236] To begin the assay, biological samples from mammary fluidcollected on nitrocellulose filters coupled with a breast pump 10, 10′as generally described above are eluted electrophoretically intophosphate buffered saline to provide a test sample, according tostandard methods. Alternatively, whole mammary fluid or other types ofbiological samples obtained from mammary fluid can be constituted in anappropriate medium or mixture to provide a test sample for the assay.Once the test sample is thus provided, it is then assayed according tothe HME-Ags radioimmunoassay (RIA) methods described in Ceriani et al.,Breast Cancer Res. Treat. 15:161-174, 1990 (incorporated herein byreference in its entirety).

[0237] Briefly, the RIA includes two preliminary treatments of thebiological samples to separate interfering factors: a centrifugationstep to separate out any fat present, and a second, precipitation stepto precipitate potential immunocomplexes using polyethyleneglycol (PEG).The next steps comprise the assay proper, where HMFG antigen bound to asolid support (microtiter plates) is presented to stoichiometric orlesser amount of the α-HME antibody probe, and binding of the α-HME iscompeted by the biological samples from mammary fluid preliminarilytreated as above. The amount of α-HME bound to HMFG antigen on the solidphase is determined in a final step by detection of the α-HME antibodyprobe by radioiodinated, affinity-purified rabbit anti-mouseimmunoglobulin.

[0238] Solutions used in the assay are as follows: i) Phosphate bufferedsaline (PBS): 176 ml 0.05M KH₂PO₄, 608 ml 0.05M Na₂HPO₄, and 8 g NaClbrought up to 1000 ml in H₂O (pH7.4). ii) RIA buffer: 0.1% BSA, in 0.3%Triton-X-100 (Research Prod. International Corp., Mount Prospect, Ill.)plus 0.05% sodium azide in PBS. iii) Detergent buffer: 0.3% Triton-X-100plus 0.05% sodium azide in PBS. iv) Buffered polyethylene glycol (PEG):6.6% PEG (M.W. 8000) (Sigma) plus 0.05% sodium azide in PBS)¹²⁵I-labeled affinity-purified rabbit anti-mouse immunoglobulin(Rα-mouse Ig) (Antibodies, Inc., Davis, Calif.), radioiodinated by thechloramine-T procedure as reported (Ceriani et al., Proc. Natl. Acad.Sci. USA 79:5420-5425, 1982) and diluted to 4×10⁶ cpm/ml, in RIA buffer.Rabbit polyclonal anti-HMFG antibodies or rabbit anti-human mammaryepithelial antibodies (α-HME) were prepared and assayed as described(Id.).

[0239] To prepare a standard curve for evaluating assay results, controlsamples from normal human mammary fluid (exposed to nitrocellulosefilters and eluted in the same manner as the nitrocellulose adsorbed,eluted test sample, or alternatively provided as normal whole mammaryfluid or other selected type of sample obtained from normal mammaryfluid, constituted in an appropriate medium or mixture to provide asuitable control assay sample) are centrifuged for 7 min at 10,240 rpmat 10° C. The upper white band formed at the top of the sample (if thereis one) is discarded. Fresh 100 μg protein/ml solution of lyophilizeddilipidated HMFG (Ceriani et al., Proc. Natl. Acad. Sci. USA 74:582-586,1977) in detergent buffer is prepared and sonicated at 10 secondintervals for a total of 4 minutes (10 sec. of sonication, followed by a10 sec. silent period) using a double step micro tip horn at 25 watts ona Sonifier Cell Disrupter 185 (Branson, Danbury, Conn.) at 4° C. HMFGsolutions at concentrations of 0, 10, 33.3, 100, 333.3, and 1000 ngprotein/ml are prepared in spun female sera, and 3 aliquots of 180 μl ofeach HMFG level in normal female sera are pipetted into 400 pipolyethylene microcentrifuge tubes (West Coast Sci. Emeryville, Calif.).150 μl of 6.6% PEG solution is added to each microcentrifuge tube, andthe tubes are incubated overnight on a rotating shaker at roomtemperature.

[0240] Test samples are processed in a comparable manner, bycentrifuging 300-350 μl of the eluted nitrocellulose filtrate insolution (or, alternatively, of mammary fluid or other assay samplealternative) in a 400 μl microcentrifuge tube for 5-7 min. at 10,240 rpmat 10° C. The microcentrifuge tubes are then cut with a razor bladebelow the white band formed by the sera (if there was one) and 180 μl ofremaining sera is transferred to a new microcentrifuge tube. 150 μl of a6.6% PEG solution is then added to each microcentrifuge tube, and thetubes are incubated overnight on a rotating shaker at room temperature.

[0241] Day two

[0242] (1) α-HME is diluted to its appropriate concentration indetergent buffer. The antibody solution has stoichiometric or lesseramounts of A-HME to 6 ng HMFG protein equivalent (prot. eq.). Six ng ofHMFG is covalently bound to microtiter plates by the methylated BSAprocedure previously described by Ceriani, in Monoclonal Antibodies andFunctional Cell Lines, pp.398-402, Kennet et al (eds), Plenum Press, NewYork, 1984, incorporated herein by reference in its entirety.

[0243] (2) To process test samples and control samples on the secondday, microcentrifuge tubes are centrifuged for 7 min. at 10,240 rpm at10° C. in a SHMT rotor with a Sorvall RC5C centrifuge. In triplicate, 55μl of supernatant is pipetted into empty microtiter plate wells(Dynatech, Alexandria, Va.), and any precipitate pelleted is leftundisturbed. 25 μl of 6.6% PEG solution is added to each well. 30 μl ofα-HME diluted in detergent buffer is also added to each well, and anon-porous Scotch® tape is placed over the wells to avoid evaporation.The microtiter plate is then incubated overnight at room temperature ona rotary shaker.

[0244] Day Three

[0245] The microtiter plates are centrifuged (3000 r.p.m.) for 30minutes at room temperature to decant suspended perceptible matter. 50μl of RIA buffer is added to wells of microtiter plates containing 6 ngHMFG and aspirated off after 5 minutes.

[0246] The total contents of microtiter plates from 1), save for anyprecipitation induced by the PEG and already pelleted, are carefullytransferred to the wells of another set of microtiter plates containing6 ng HMFG per well (Day 2,1), above.

[0247] The microtiter plates are incubated for 3 hours with rotatingagitation at room temperature. The plates are washed 5 times with RIAbuffer using Dynadrop SR4 automatic dispenser form Dynatech. 50 μl ofthe radioiodinated affinity-purified rabbit anti-mouse immunoglobulindiluted in RIA buffer is then adder per well. The plate is covered withtape and incubated with rotating agitation for 2 hours at roomtemperature. The plate is washed 5 times with RIA buffer. Wells are cutand counted in a gamma counter.

[0248] The results of these assays will yield important informationconcerning the presence and/or status of cancer in patients, comparablein scope and value to the data provided by serum assays conducted forthe HME-Ag breast cancer marker by Ceriani et al, Breast Cancer Res.Treat. 15:161-174, 1990. By selecting patient and control samples anddeveloping and evaluating comparative data according to the proceduresfollowed by Ceriani and his coworkers, the assay methods of theinvention will also be readly adapted for use in direct clinicalapplications to determine both prognostic and treatment relatedvariables in breast cancer patients. Reagents and conditions for theassays can of course be substituted or adjusted depending on a varietyof anticipated variables, by applying well known immunological methodsand principles.

EXAMPLE 6 Competitive Radioimmunoassay for Non-Penetrating Glycoproteinin Biological Samples From Mammary Fluid

[0249] This competitive radioimmunoassay is based on the displacementbybreast epithelial antigens contained in biological samples frommammary fluid obtained according to the methods of the invention of thebinding of stoichiometric or lesser quantities of the monoclonalantibody Mc5 to a solid-phase-bound antigen known as non-penetratingglycoprotein (NPGP) contained in HMFG. HMFG is bound to a solid supportand exposed to the Mc5 antibody during an incubation period allowing theantibody to bind the NPGP antigen in solid phase-bound HMFG. Thepresence and/or level of NPGP in the biological sample is ultimatelyexamined by ability of the sample to compete for Mc5 binding to the NPGPantigen in the solid phase-bound HMFG, as detected and/or measured usinga radiolabeled goat anti-mouse antibody to bind and label the Mc5antibody probe.

[0250] Buffer and other solutions and reagents in this example aregenerally the same as those used for the HME-Ags polyclonal antibodyradioimmunoassay described in Example 4, above. To provide test samplesfor the assay, biological samples from mammary fluid contained onnitrocellulose filters are eluted electrophoretically into phosphatebuffered saline, according to standard methods. Alternatively, wholemammary fluid or other types of biological samples obtained from mammaryfluid can be constituted in an appropriate medium or mixture to providea test sample for the assay. Once the test sample is thus provided, itis then assayed according to the NPGP/Mc5 radioimmunoassay (RIA) methodsdescribed in Ceriani et al., Breast Cancer Res. Treat. 15:161-174, 1990(incorporated herein by reference in its entirety), as follows:

[0251] 400 μl of pooled normal female mammary fluid (exposed tonitrocellulose filters and eluted in the same manner as thenitrocellulose adsorbed, eluted test sample, or alternatively providedas normal whole mammary fluid or other types of biological samplesobtained from normal mammary fluid constituted in an appropriate mediumor mixture to provide a test sample) to provide a suitable controlsample, which is diluted to 2.4 ml using RIA buffer at a 1:6concentration.

[0252] A 500 μg/ml solution of lypholized HMFG is prepared in 1× PBSwith 0.3% Triton-X-100, 0.05% sodium azide, and sonicated using a doublestep micro tip horn at 25 watts on a Sonifier Cell Disrupter 185(Branson, Danbury, Conn.) for 4 minutes (10 sec. sonication, 10 sec.silent period, at 4° C.).

[0253] Solutions to prepare a standard curve are prepared using the 2.4ml 1:6 normal female serum and increasing amounts of HMFG (0, 0.25, 2.5,25, 50 μg/ml HMFF, as described above in Example 4).

[0254] Each test assay sample is diluted 1:6 with RIA buffer (40 μL ofserum to 200 μL RIA buffer) to form a diluted test assay sample, andvortexed.

[0255] Mc5 stock solution is prepared so that it contains less thanstoichiometric amounts of antibody to 100 ng protein/well of HMFGcovalently bound to microtiter plates prepared as previously describedby Ceriani, in Monoclonal Antibodies and Functional Cell Lines, pp.398-402, Kennet et al. (eds), Plenum Press, New York, 1984, incorporatedherein by reference in its entirety

[0256] 200 μl RIA buffer are added to each well of 100 ng HMFG and thenaspirated after 5 minutes.

[0257] To prepare a standard curve, 30 μl of HMFG standardizingsolutions (as in 3 above) are added in quadruplicate to a 100 ngprotein/well HMFG microtiter well. 30 μl of diluted test assay sample(or, alternatively, of mammary fluid or other assay sample alternative)are added in triplicate to 100 ng/well HMFG microtiter wells.

[0258] To each well 20 μl of the Mc5 stock solution is added.

[0259] Microtiter plates are covered with nonporous Scotch® tape andincubated overnight at room temperature on a rotating agitator.

[0260] The next day the wells are aspirated and washed 5 times with RIAbuffer.

[0261] To each well 50 μL of 200,000 cpm/50 μl ¹²⁵i-goat anti-mouseantibody are dispensed. The wells are covered with nonporous tape andplaced on a rotating agitator for 3 hours at room temperature.

[0262] Wells are washed 5 times with RIA buffer.

[0263] Each well is cut and the radioactivity is counted using a gammacounter.

[0264] The results of these assays will yield important informationconcerning the presence and/or status of cancer in patients, comparablein scope and value to the data provided by serum assays conducted forthe NPGP breast cancer marker by Cerianiet al., Breast Cancer Res.Treat. 15:161-174, 1990. By selecting patient and control samples anddeveloping and evaluating data according to the well known proceduresfollowed by Ceriani and his coworkers, the assay methods of theinvention will be readily adapted for use in direct clinicalapplications to determine both prognostic and treatment relatedvariables in breast cancer patients. As will be understood by thoseskilled in the art, reagents and conditions for the assays can besubstituted or adjusted depending on a variety of anticipated variables,according to well known immunological methods and principles.

EXAMPLE 7 Solid Phase Immunoassay for Mucinous Carcinoma AssociatedAntigen in Mammary Fluid

[0265] This example uses a sensitive, solid phase immunoassay to detectthe mucinous carcinoma associated antigen (MCA) in biological samplesfrom mammary fluid obtained according to the methods of the invention.MCA concentrations are determined using an antibody-bead immunoassay kitprovided by Hoffinan-La Roche (Basel, Switzerland), and using thereagents and procedures provided by the manufacturer and described infurther detail in Eskelinen et al., Anticancer Res. 9:437-440, 1989.Briefly, test assay samples of whole mammary fluid and standards arefirst incubated with MCA monoclonal antibody beads and then, afterappropriate washings, enzyme (horseradish peroxidase) labeled secondaryantibody is added. During the second incubation the anti-MCA enzymeconjugates are attached to the antibody antigen complex on the beads.Excess conjugates are removed by washing and, finally, enzyme substrateare added and the color formed is recorded.

[0266] The solid phase assay format presented in this example can beadapted for use in a wide array of other assays to detect and/or measureother cancer markers besides the MCA marker, with enhanced sensitivity.In addition, the results of these assays can be evaluated along withthose of complementary assays detecting and/or measuring differentmarkers to yield more precise information concerning the presence and/orstatus of cancer in patients, as exemplified by the combinatorial MCA/CA15-3 assays described by Eskelinenet al., Anticancer Res. 9:437-440,1989; see also Eskelinen et al., Anticancer Res. 8:665-668, 1988, eachincorporated herein by reference in its entirety.

EXAMPLE 8 Western Analysis of Proteins From Cellular Fractions of HumanMammary Fluid Using Polyclonal and Monoclonal Antibody Probes to DetectVasopressin

[0267] A variety of assays are provided by the present invention thatfocus on cellular samples from human mammary fluid. In general, theseassays rely on isolation by standard separation methods (e.g.centrifugation, sucrose gradient, etc.) of cells, membranes or othercell components from whole mammary fluid expressed according to theabove methods. Biological samples containing whole cells from expressedmammary fluid are particularly useful for cytological and cytochemicalexamination to detect and evaluate breast cancer in patients. Biologicalsamples containing purified cell membrane fractions from human mammaryfluid are particularly useful in this context, for example to detectand/or measure breast cancer markers that are expressed byalveolar-ductal cells as intracellular or membrane bound proteins andare therefore not as readily detected in liquid fractions of mammaryfluid as secreted proteins.

[0268] The present example focuses on assays for detecting the peptidehormone vasopressin in biological samples from mammary fluid, usingmethods adapted from North et al, Breast Cancer Res. Treat. 34:229-235,1995. Specifically, this assay uses a test sample of crude proteinisolated from a pooled sample of cells obtained from expressed mammaryfluid. The cells are separated from whole mammary fluid according tostandard methods, and crude protein is extracted from the cells bysonication in 100 volumes of 0.1 M HCl. The resuling protein suspensionsare then centrifuged at 1500× g for 10 min. at ambient temperature, andsoluble protein is precipitated with 40% TCA. This protein is pelletedby centrifugation at 10,000× g for 2 min. TCA is then removed frompellets by washing (×2) with ether. Protein is resuspended in 0.1 M TrisHCl (pH8.7), reduced with mercaptoethanol at 100° C. for 5 min. (and insome cases S-alkylated with N-ethyl maleimide), and subjected toSDS-PAGE electrophoresis on 15% gels at pH 9.3 using the method ofLaemeli, Nature 227:680-685, 1970, incorporated herein by reference inits entirety. Separated proteins are then electrophoreticallytransferred with 20 mM Tris glycine (pH 8.0) to Immobilon PVDF membranes(Millipore, Bedford, Mass.). These membranes are blocked with a 5%non-fat milk solution, washed (1×15 min., 2×5 min.) with PBS containing0.5% Triton, and incubated with preparations of mouse monoclonalantibody to VP-HNP, with rabbit polyclonal antibodies to VP, with rabbitpolyclonal antibodies to VAG, or with ubiquitous mouse or rabbit IgG(negative controls) (for description of antibodies and antibodypreparation see North et al., Breast Cancer Res. Treat. 34:229-235,1995, incorporated herein by reference in its entirety), for 1 h atambient temperature. Following a second wash in PBS-Triton (1×15 min.,2×5 min.), the membranes are treated, respectively, with goat anti-mouseIgG-horseradish peroxidase conjugate or goat anti-rabbit IgG-horseradishperoxidase conjugate for 1 h, and then washed with PBS-Triton (1×15min., 4×5 min.). Immunoreactive proteins are visualized using an ECLWestern Blotting Detection System with exposure of x-ray film from 10seconds to 5 min. Prestained SDS-PAGE standard proteins are employed asmolecular size markers.

[0269] Recent studies suggest that vasopressin is universally expressedin breast carcinoma and is absent from normal breast cells. North etal., Breast Cancer Res. Treat. 34:229-235, 1995. These and other resultsindicate that vasopressin and its relatives are important breast cancermarkers that can be readily detected using immunological assays ofproteins isolated from breast tumor cells. Accordingly, the results ofthe present example using cell samples isolated from human mammary fluidare also expected to yield important information concerning the presenceand/or status of cancer in patients.

EXAMPLE 9 Quantification of Carcinoembryonic Antigen in BiologicalSamples From Mammary Fluid by Dot Immunoblotting Assay

[0270] Among the more sensitive assays of the invention, useful formeasuring low levels of breast cancer markers and for detecting markerswhen only small volumes of expressed mammary fluid are available, is thedot immunoblotting assay. In the present example, carcinoembryonicantigen (CEA) is measured in whole mammary fluid using an Elmotechanti-CEA monoclonal antibody kit (Mochid Pharmaceutical Co., Tokyo,Japan) in a dot blot assay format. Briefly, anti-CEA monoclonal antibodyis diluted to appropriate concentrations and coated on the plastic film.Aliquots (5 μl) of either standard CEA solution (0, 100, 200, and 500ng/ml), or of the whole mammary fluid assay sample, are smeared on theimmobilized film. Assay standards are prepared from purified antigenpreparations, in accordance with the Elmotec kit manufacturer'sinstructions. If necessary, 1000 ng/ml CEA solution is also used as astandard. After drying at room temperature, the film is exposed toperoxidase-conjugated anti-CEA antibody for 20 min at room temperature.The film is then washed extensively with 1 M saline containing 0.5%(v/v) Tween 20. The enzyme reaction is visualized usingtetramethylbenzidine as a chromogen. The developing solution consists of0.05 mM tetramethylbenzidine and 0.01% hydrogen peroxide in Mcllvainbuffer (0.1% M phosphate-citrate buffer), pH 5.0, containing 10%methanol. The concentration of CEA in the mammary fluid assay sample isdetermined by comparing the color intensities with a correspondingstandard.

[0271] The assay disclosed in the present exanple, and related assaysincorporating antibodies to other tumor markers besides CEA, areparticularly useful for measuring low levels of breast cancer markersand for detecting markers in limited sample volumes. The results ofthese assays will yield important information to determine bothprognostic and treatment related variables in breast cancer patients. Aswill be understood by those skilled in the art, reagents and conditionsfor the assays can be substituted or adjusted depending on a variety ofanticipated variables, according to well known immunological methods andprinciples.

EXAMPLE 10 Detection of Procathepsin D and Cathepsin D Activity inBiological Samples From Mammary Fluid

[0272] Cathepsin D is a lysosomal aspartic proteinase that has beenstudied intensively as a marker for cancer processes necessary formetastasis. In the present example, polyclonal antibodies againstprocathepsin D are used to immunoprecipitate and immunochemically detectproteins from whole mammary fluid or cell lysates from mammary fluid,generally according to the methods disclosed in Vetvicka et al.,Biochem. Mol. Biol. Int'l. 30:921-928, 1993 (incorporated herein byreference in its entirety). Alternatively, or as a complementary assay,the protease activity of cathepsin D is detected, also according to themethods disclosed in Vetvicka et al. (Id.). Briefly, pooled wholemammary fluid (preferably 3 ml if available) is diluted with 3 ml ofbuffer A (50 mM Tris.HCl, 5 mM CaCl₂, 1 mM MgCl₂, 500 mM NaCl pH 7.2).The suspension is centrifuged for 30 minutes at 10,000 g. The resultingwater phase is centrifuged again under the same conditions. The solublepart (total of approximately 4.5 ml) is loaded on a 1 ml column ofConcanavalin A Sepharose (Pharmacia, Uppsala, Sweden) equilibrated inbuffer A, and after washing with buffer A the retained proteins areeluted using 0.75 M methyl a-D-mannopyranoside. The fractions (250 μl)are analyzed for cathepsin D activity using the ¹⁴C hemoglobin assay asdescribed by Lin et al., J. Biol. Chem. 264:4482-4489, 1989(incorporated herein by reference in its entirety), by western blots andby silver-stained electrophoresis. The inhibition of human milkprocathepsin D is accomplished by adding 2 μl of 1 mM pepstatin A(Boehringer Manhein, Germany) dissolved in methanol to the reactionmixture.

[0273] This assay provides but one example of many possible embodimentsof the invention that incorporate known biochemical assays, in additionto, or supplemental to immunological assays, to evaluate biologicalsamples from mammary fluid to determine cancer related variables. Thefundamental methods provided herein for obtaining samples from humanmammary fluid render these assays readily adaptable for widespreadclinical application to detect and/or measure the activity of a subjectbreast cancer marker within a non-invasive screening protocol.

[0274] Those with ordinary skill in the art will appreciate that otherembodiments and variations of the invention are possible which employthe same inventive concept described above. Most particularly, a wideand rapidly expanding array of useful breast cancer markers (includingproteins, DNA and RNA sequences and other markers) and probes (includingimmunological, nucleotide and biochemical probes) are readily availablefor adaptation and use within the methods and kits of the invention.These markers and probes are described or referenced to a large extentin the literature cited and incorporated within the present disclosure,or are elsewhere published in the literature or well known in the art.Among these known and emerging markers and probes, useful exampleswithin the invention include Her 2 (also known as erbB-2 and neu). Her 2is a transmembrane glycoprotein growth factor receptor of the EGFreceptor family encoded by a gene located on chromosome 17q, a region offrequent amplification in breast cancer cell lines. This marker ishighly predictive of breast cancer and can be detected in cellularsamples of the invention using known nucleotide probes to detect geneticdefects in Her 2, or to detect and/or measure mRNA to determineoverexpression of Her 2 linked to increased proliferation of cancercells. (See for example, Visscher et al., In Weinstein and Graham (eds)Advances in Pathology and Laboratory Medicine, vol. 5, St. Louis, MosbyYuear Book, 1992, pp. 123-161; Barbareschiet al., Am. J. Clin. Pathol.98:408-418, 1992; Slamonetal., Science 235:177-182, 1987; eachincorporated herein by reference in its entirety). Protein levels of Her2 are also readily detected using available immunological probes. (Forreview see Porter-Jordan et al, Hematol. Oncol. Clin. North Amer.8:73-100, 1994 and articles cited on page 80 therein, each incorporatedherein by reference in its entirety). Additional markers for use withinthe invention include EGF and the EGF receptor, for which immunologicaland non-immunological probes and assay methods readily adaptable withinthe invention are characterized in detail at page 80-81 of Porter-Jordanet al., Hematol. Oncol. Clin. North Amer. 8:73-100, 1994 and in thereferences cited therein, each incorporated herein by reference in itsentirety. Additional examples of proliferation markers, growth factorsand receptors, proteases, adhesion factors, angiogenic factors,oncogenes and tumor suppressor genes that provide useful breast diseasemarkers and probes within the methods and kits of the invention includeKi67 Growth Factor, Cyclin D1, Proliferating Cell Nuclear Antigen,Transforming Growth Factor, Tissue Plasminogen Activator, Insulin GrowthFactor Receptors, Collagenase Type IV, Laminin Receptor, Integrins, p53,rb, nm23, ras, c-myc,

myb, Heat Shock Proteins, Prolactin, Neuron-Specific Enolase, IR-14, KA1, KA 14, AlphaLactalbumin, Actin, IL-10, S-100 protein, Vimentin,Epithelial Membrane Antigen, bc1-2, CA15-3, CA 19-9, Tn Antigen,Alpha-lactalbumin, LASA, Gal-GalNAC, GCDFP-15, Le(y)-RelatedCarbohydrate Antigen, CA 125, uPA, uPA related antigens and complexes,uPA Receptor, PA1-1 and PA1-2, Beta-glucuronidase, CD31, CD44 splicevariants, blood group antigens including ABH, Lewis, and MN, and geneticlesions or altered expression levels of CCND1, EMS1, BRCA1 and BRCA2genes, and many others, for which immunological and non-immunologicalbinding partners, probes and assay methods are known and readilyadaptable within the invention.

[0275] Although the foregoing invention has been described in detail byway of example for purposes of clarity of understanding, it will beapparent to the artisan that certain changes and modifications arecomprehended by the disclosure and may be practiced without undueexperimentation within the scope of the appended claims, which arepresented by way of illustration not limitation.

What is claimed is:
 1. A method for handling or processing a biologicalsample of mammary fluid or a component thereof for use in a diagnosticassay to detect or quantify a breast disease marker in said samplecomprising the step of: providing or obtaining said biological sample ofmammary fluid or component thereof in a fluid-retaining reservoir havinga top end defining a primary opening for access to the sample and anouter reservoir wall defining one or more air ports that communicatebetween the outer wall and an inner lumen of the vial.
 2. The methodaccording to claim 1, wherein the fluid-containing reservoir memberfurther comprises closure means for closure of the reservoir after saidsample is introduced therein to prevent sample contamination andspillage.
 3. The method according to claim 2, wherein the closure meanscomprises a cap adapted to sealably engage a top end of the removablereservoir member.
 4. The method according to claim 2, wherein theclosure means further comprises secondary closure means to sealablyclose the air port(s) after said sample is introduced into saidreservoir.
 5. The method according to claim 4, wherein said secondaryclosure means comprise an adhesive seal or sticker sized and constructedto adhere to an outer wall of the reservoir member surrounding an airport opening.
 6. The method according to claim 4, wherein said secondaryclosure means comprises a combined closure and labeling device whichfunctions as a secondary closure mechanism to seal the air port(s) ofthe removable reservoir and as a labeling template to provide a writingsurface for sample labeling.
 7. The method according to claim 4, whereinsaid secondary closure means comprises a combined closure and labelingtab or sticker which may be directly applied to seal the air port aftersample collection having a first, closure-forming surface forapplication over the air port to form a seal by juxtaposition oradhesive contact with an outer wall of the removable reservoir, and asecond, labeling surface opposite the closure-forming surface made of ablank template material suitable for receiving a stable, ink or graphiteimprint thereon.
 8. The method according to claim 7, wherein said first,closure-forming surface bears an adhesive coating resistant todisruption by contact with aqueous solutions.
 9. The method according toclaim 4, wherein said secondary closure means comprises a combinedclosure and labeling tab or sticker which is pre-attached to theremovable reservoir member in a first, open configuration and can bemanually repositioned or otherwise manipulated after sample collectionto a second, closed configuration to form a seal or closure against theair port(s).
 10. The method according to claim 4, wherein said secondaryclosure means comprises an adhesive tab or strip folded in the openconfiguration to form an inner layer affixed to the reservoir proximateto the air port and an outer layer folded over the inner layer, saidouter layer providing the first, closure-forming surface and the second,labeling surface, wherein the outer layer can be unfolded away from theinner layer and wrapped around the reservoir so that the closure-formingsurface covers the air port to form a fluid-resistant closure and thelabeling surface faces outward for recordation of sample data.
 11. Themethod according to claim 10, wherein the outer layer is optionallysecured in a folded-back position against the inner layer by adhesiveengagement of the labeling surface with the inner layer.
 12. The methodaccording to claim 11, wherein said first, closure-forming surface bearsan adhesive coating that is protected in the open configuration byfolding of an end segment of the outer layer bearing the adhesivecoating back, so that the closure forming surface provides a protectivesurface to shield the adhesive prior to closure, whereby the end segmentcan be lifted and pulled outward to unfold the end segment to separatethe adhesive coating on the closure-forming surface from the protectivesurface and to release the outer layer from the inner layer for closingof the air port(s).
 13. The method according to claim 1, wherein thefluid-retaining reservoir is a modified cytology vial.
 14. The methodaccording to claim 1, wherein the fluid-retaining reservoir comprises aremovable fluid reservoir member of a sample collection housing of amammary fluid collection device.
 15. The method according to claim 14,wherein the fluid-retaining reservoir is a rigid sample collection tubeor vial removably connected with an outer casing member of a samplecollection housing of said collection device.
 16. The method accordingto claim 15, wherein the fluid-retaining reservoir is adapted forremovable, sealable connection with said outer casing member of saidhousing to form an airtight coupling therewith.
 17. The method accordingto claim 16, wherein the fluid-retaining reservoir is a cytology vialsealably connectable with said outer casing member to form an airtightcoupling therewith.
 18. The method according to claim 17, wherein thefluid-retaining reservoir can be removably nested within said casingmember to form a substantially airtight contact between an inner wall ofthe casing member wall and an outer wall, or a top or bottom end, of thereservoir member.
 19. The method according to claim 1, wherein an outerwall of the fluid-retaining reservoir bears a circumferential ridge, finor O-ring.
 20. The method according to claim 19, wherein thefluid-retaining reservoir comprises a removable fluid reservoir memberof a sample collection housing of a mammary fluid collection device andthe circumferential ridge, fin or O-ring is adapted to engage and make acircumferential airtight seal against an inner wall of a casing memberof a sample collection housing of said device.
 21. The method accordingto claim 1, wherein said mammary fluid sample or component thereofincludes whole cells or cell fragments.
 22. The method according toclaim 1, further comprising the step of accessing said sample withinsaid reservoir to transfer or process the sample for detection orquantification of said breast disease marker.
 23. The method accordingto claim 22, further comprising the step of transferring said sample toa second reservoir or other sample container or template for processingthe sample to detect or quantify said breast disease marker.
 24. Themethod according to claim 23, wherein said step of processing saidsample to detect or quantify said breast disease marker comprisesstaining cells or cell fragments from said sample with a cytologicalstain to detect a cytological marker.
 25. The method according to claim22, further comprising the step of processing said sample to detect orquantify said breast disease marker by microscopic examination ofstained cells or cell fragments from said sample.
 26. The methodaccording to claim 1, wherein said biological sample includes one ormore components selected from the group consisting of whole mammaryfluid, whole cells, cell fragments, cell membranes, purified proteins,bulk proteins, glycoproteins, peptides and polynucleotide components ofa primary mammary fluid sample.
 27. The method according to claim 1,wherein the breast disease marker is a breast cancer marker.
 28. Themethod according to claim 1, wherein the breast disease marker isselected from the group consisting of a protein, a peptide, aglycoprotein, a lipid, a DNA polynucleotide and an RNA polynucleotide.29. The method according to claim 1, wherein the breast disease markeris selected from the group consisting of Ki67 Growth Factor, Cyclin D1,Proliferating Cell Nuclear Antigen, Transforming Growth Factor TissuePlasminogen Activator, Insulin Growth Factor Receptors, Collagenase TypeIV, Laminin Receptor, Integrins, p53, rb, nm23, ras, c-myc, Heat ShockProteins, Prolactin, Neuron-Specific Enolase, IR-14, KA 1, KA 14,Alpha-Lactalbumin and Actin.
 30. The method according to claim 1,wherein the breast disease marker is selected from the group consistingof CEA, HMFG, MCA, vasopressin and cathepsin.
 31. The method accordingto claim 1, further comprising the step of detecting or quantifying saidbreast disease marker by ELISA immunoassay, immunoprecipitation assay,or solid phase immunoassay.
 32. The method according to claim 1, whereinsaid biological sample comprises an oxytocin-induced sample of mammaryfluid or a component thereof
 33. The method according to claim 32,wherein said biological sample comprises whole cells or cell fragmentsfrom said oxytocin-induced sample of mammary fluid.
 34. A mammary fluidcollection reservoir for collection, handling and processing biologicalsamples of mammary fluid comprising: a rigid tube or vial having a topend defining a primary opening for collecting and accessing the mammaryfluid sample and a cylindrical outer wall defining a reservoir lumenclosed at a bottom end thereof, said sidewall defining one or more airports that communicate between the outer wall and the lumen of the vial.35. The mammary fluid collection reservoir of claim 34, wherein thereservoir comprises a removable fluid reservoir member of a samplecollection housing of a mammary fluid collection device.
 36. The mammaryfluid collection reservoir of claim 35, wherein the reservoir comprisesa rigid sample collection tube or vial removably connected with an outercasing member of the sample collection housing of the mammary fluidcollection device.
 37. The mammary fluid collection reservoir of claim36, wherein the reservoir is adapted for removable, sealable connectionwith said outer casing member of the housing, to form an airtightcoupling therewith.
 38. The mammary fluid collection reservoir of claim37, wherein the reservoir is a cytology vial sealably connectable withthe outer casing member to form the airtight coupling.
 39. The mammaryfluid collection reservoir of claim 35, wherein the reservoir can beremovably nested within an outer casing member of the sample collectionhousing to form a substantially airtight contact between an inner wallof the casing member and an outer wall or top or bottom end of thereservoir member.
 40. The mammary fluid collection reservoir of claim35, wherein the outer wall of the reservoir is provided with acircumferential ridge, fin or O-ring.
 41. The mammary fluid collectionreservoir of claim 34, further comprising closure means for closing thereservoir after a sample of mammary fluid is introduced therein toprevent sample contamination and spillage.
 42. The mammary fluidcollection reservoir of claim 41, wherein said closure means comprises acap adapted to sealably engage the top end of the reservoir and sealablyclose said primary opening.
 43. The mammary fluid collection reservoirof claim 41, wherein said closure means includes secondary closure meansadapted for closure of said one or more air port(s).
 44. The mammaryfluid collection reservoir of claim 43, wherein said closure meansincludes a secondary closure device selected from a plug, cap oradhesive cover sized and constructed to engage or adhere to the outerwall of the reservoir member at or surrounding the air port opening(s)to form a secondary closure.
 45. The mammary fluid collection reservoirof claim 41, wherein said secondary closure means comprise a combinedclosure and labeling device which functions as a secondary closuremechanism to seal the air port(s) of the reservoir and as a labelingtemplate to provide a writing surface for sample labeling.
 46. Themammary fluid collection reservoir of claim 45, wherein said secondaryclosure means comprise a combined closure and labeling tab or stickerfor application to the outer wall of the reservoir to seal the airport(s) after the sample is collected.
 47. The mammary fluid collectionreservoir of claim 46, wherein said tab or sticker has a first,closure-forming surface for application over the air port(s) to form aseal by juxtaposition or adhesive contact with the outer wall of thereservoir, and a second, labeling surface opposite the closure-formingsurface made of a blank template material for imprinting writteninformation thereon.
 48. The mammary fluid collection reservoir of claim45, wherein said secondary closure means is pre-attached to theremovable reservoir member in a first, open configuration and can bemanually repositioned or otherwise manipulated after sample collectionto a second, closed configuration to form a seal or closure against theair port(s) and provide and exposed writing surface for sample labeling.